Gn1a - Cytokinin Oxidase Regulates Rice Grain Production(细胞色素氧化酶对水稻产量的调控)

GNAO1与胃癌预后的关系及其分子机制研究的开题报告题目：GNAO1与胃癌预后的关系及其分子机制研究一、研究背景胃癌是全球范围内较为严重的恶性肿瘤之一，其发病率和死亡率排名第五和第三。

尽管近年来在胃癌的诊断和治疗方面取得了一定的进展，但仍然存在一定的治疗难度和预后不良等问题。

因此，对胃癌相关的分子机制进行深入研究，以发现新的治疗手段、提高患者的预后，具有非常重要的意义。

GNAO1是一种G蛋白α亚基，在多种人类癌症中都表达有所改变。

已发现GNAO1在某些白血病、肝癌等多种人类癌症中的超表达会导致不良预后，但其在胃癌中的作用还不清楚。

因此，需要对GNAO1在胃癌中的表达与预后的关系及其分子机制进行深入研究。

二、研究目的本研究旨在：1）探究GNAO1在胃癌患者中的表达情况及其与临床病理特征和预后的关系；2）分析GNAO1对胃癌细胞增殖、侵袭和凋亡的影响，并探究其作用机制。

三、研究内容和方法1）对30例胃癌患者的癌组织和对应的癌旁组织进行GNAO1的免疫组化（IHC）染色，评价其在胃癌组织中的表达情况，并分析其与临床病理特征和预后的关系。

2）构建GNAO1高表达和低表达的胃癌细胞株，并分别进行流式细胞术和MTT实验，评价GNAO1对胃癌细胞凋亡和增殖的影响。

3）利用Transwell侵袭实验和Wound Healing实验，研究GNAO1对胃癌细胞侵袭和迁移的影响；同时使用Western blotting和Real-time PCR等方法，探究GNAO1对胃癌细胞凋亡和增殖的分子机制。

四、研究意义通过本研究的探究，可以深入了解GNAO1在胃癌中的作用机制，为胃癌的临床诊断和治疗提供理论支持。

同时，为胃癌的治疗手段开发和患者预后改善提供有价值的参考。

Theor Appl GenetDOI 10.1007/s00122-008-0711-9ORIGINAL PAPERFine mapping of a major quantitative trait loci, qSSP7, controlling the number of spikelets per panicle as a single Mendelian factor in riceY. Z. Xing · W. J. Tang · W. Y. Xue · C. G. Xu ·Qifa ZhangReceived: 29 June 2007 / Accepted: 8 January 2008© Springer-Verlag 2008Abstract In our previous studies, one putative QTL a V ecting number of spikelets per panicle (SPP) was identi-W ed in the pericentromeric region of rice chromosome 7 using a recombinant inbred population. In order to de W ne the QTL (qSPP7), RI50, a recombinant inbred line with 70% of genetic background same as the female parent of Zhenshan 97, was selected to produce near-isogenic lines for the target region in the present study. In a BC2F2 popu-lation consisting of 190 plants, the frequency distribution of SPP was shown to be discontinuous and followed the expected Mendelian ratios (1:2:1 by progeny test) for single locus segregation. qSPP7 was mapped to a 0.4cM region between SSR marker RM3859 and RFLP marker C39 based on tests of the BC2F2 population and its progeny. Its additive and dominant e V ects on SPP were 51.1 and 24.9 spikelets, respectively. Of great interest, the QTL region also had e V ects on grain yield per plant (YD), 1,000 grain weight (GW), tillers per plant (TPP) and seed setting ratio (SR). Signi W cant correlations were observed between SPP and YD (r=0.66) and between SPP and SR (r=¡0.29) in the progeny test. 1082 extremely small panicle plants of a BC3F2 population containing 8,400 individuals were further used to W ne map the QTL. It turns out that qSPP7 co-segre-gated with two markers, RM5436 and RM5499 spanning a physical distance of 912.4kb. Overall results suggested that recombination suppression occurred in the region and posi-tional cloning strategy is infeasible for qSPP7 isolation. The higher grain yield of Minghui 63 homozygote as com-pared to the heterozygote suggested that Minghui 63 homo-zygote at qSPP7 in hybrid rice could further improve its yield.IntroductionWith the advent of DNA molecular markers, QTL mapping has become a routine strategy for the discovery of genes involved in complex quantitative traits. Thousands of QTL have been mapped for important agronomical traits in rice (/db/cmap/map_set_info?species_ acc=rice&map_type_acc=qtl). Although primary mapping populations including F2, recombinant inbred lines (RILs) and doubled haploid lines (DHs) have been widely used for QTL mapping in rice (Li et al. 1995; Yu et al. 1997; Xu et al 2004; Fan et al. 2005; Marri et al. 2005), QTL can only be localized to a genomic region (con W dential region) rather than a locus in those populations. Following the pri-mary mapping, advanced populations such as near isogenic lines (NIL) and chromosome segment substitution lines (CSSL) can be used to map QTL to a locus as a Mendalian factor by blocking the genetic background noise (Lin et al. 2002; Zhang et al. 2006; Wang et al. 2006). Based on this strategy, several QTLs have been isolated in tomato (Frary et al. 2000) and rice (Yano et al. 2000; Takahashi et al. 2001; Li et al. 2003) in recent years. The common aspect in all the QTL cloning work is to exploit high quality NILs as advanced mapping populations.Food shortage is one of the most serious global prob-lems. The United Nations Food and Agricultural Organiza-tion (FAO) estimates that 852 million people worldwideCommunicated by A. Paterson.Y. Z. Xing and W. J. Tang contributed equally to this work. Y. Z. Xing (&) · W. J. Tang · W. Y. Xue · C. G. Xu · Q. Zhang National Key Laboratory of Crop Genetic Improvementand National Center of Plant Gene Research,Huazhong Agricultural University, Wuhan 430070, Chinae-mail: yzhxing@; yzxing@Theor Appl Genetwere undernourished between 2000 and 2002 (http://www. /documents/show_cdr.asp?url_file0/docrep/007/y56 50e/y5650e00.htm). Rice is a staple food for many coun-tries. As the world population increases, rice production has to be raised by at least 70% over the next three decades to meet growing demands. In the long run, development of high yield varieties is one of the most important goals in rice cultivation. Unfortunately, yield improvement e Y ciency is deemed to be very low due to its complex property a V ected by number of spikelets per panicle (SPP), 1,000-grain weight (GW) and tillers per plant (TPP). Of these factors, SPP was shown to be highly correlated with yield and acts as a crucial component in determining rice yield (Hua et al. 2002). Therefore, dissection of its genetic basis would be of great value in breeding high yield variety. So far, QTLs for SPP have been mapped in lots of popula-tions, which were derived from inter-subspeci W c and intra-speci W c crosses (Li et al. 1997; Yu et al. 1997; Xing et al. 2002). Although many QTL controlling SPP were mapped in rice, only Gn1a, has been cloned. Gn1a, controlling grain productivity in rice, was elucidated to be a gene encoding cytokinin oxidase/dehydrogenase (OsCKX2), an enzyme that degrades the phytohormone cytokinin. Reduced expression of OsCKX2 causes cytokinin accumu-lation in in X orescence meristems and increases the number of reproductive organs, resulting in enhanced grain yield (Ashikari et al. 2005).In our previous studies, a QTL a V ecting the number of spikelets per panicle was repeatedly mapped to one pericen-tromeric region X anked by RFLP markers C1023 and R1440 on rice chromosome 7 in several primary mapping popula-tions (Yu et al. 1997, 2002; Xing et al. 2001, 2002). In the present study, we aimed to precisely estimate its genetic e V ect under near isogenic background and W ne map the QTL as a single Mendelian factor based on a large number of NILs. Materials and methodsDevelopment of NILIn our previous study, one QTL, qSPP7, was mapped to the interval between R1440 and C1023 on chromosome 7 using a recombinant inbred population derived from the cross between Zhenshan 97 and Minghui 63 (Xing et al. 2002). The recombinant inbred line 50 (RI50), was selected to backcross with the female parent Zhenshan 97 and a BC1F3 population was obtained with two sel W ngs. RI50 was homo-zygous for Minghui 63 alleles in the targeted QTL region on chromosome 7. Moreover, approximately 70 percent of RI50 genetic background was the same as that of Zhenshan 97 based on 221 polymorphic genome-wide markers (Xing et al. 2001). One line of BC1F3 population (BC1F3-120)with homozygous Minghui 63 alleles, showing uniform number of SPP, was selected to continue backcrossing with Zhenshan 97 to produce BC2F2 and BC2F3 populations. According to the genotypes of the X anking markers, one BC2F2 plant (BC2F2-SPP7) with homozygous Minghui 63 regions surrounding qSPP7 was chosen to backcross with Zhenshan 97 to produce the BC3F1. In order to screen the genetic background of BC2F1, 150 SSR markers evenly dis-tributed on 12 chromosomes were selected from all poly-morphic markers between Zhenshan 97 and Minghui 63 (McCouch et al. 2002). Eight chromosome fragments cov-ering about 150cM on chromosomes 1, 3, 5, 6, 7, 9 and 12 (two segments), were heterozygote in BC2F1. The markers situated in these 8 fragments were further used to screen several BC3F1 plants. Finally, the one with the least back-ground markers, that is, only three small regions on chro-mosomes 5, 9 and 12 were heterozygote besides about 40cM region containing the targeted QTL on chromosome 7 (Fig.1), was selected for BC3F2 production. The popula-tions of 190 BC2F2 and BC3F2 individuals were used to evaluate the gene e V ects, respectively. The BC3F1 plant was divided into several clones at seedling stage in order to harvest amounts of selfed seeds. 8,400 BC3F2 plants were used for W ne mapping the genes.Field trial and trait measurementThe 190 BC2F2 plants with RI50, Zhenshan 97 and Minghui 63 were sown on 17 May 2002 and transplanted to the W eld Fig.1The graphic genotype of the BC3F1 plant. The black and white chromosome segments were heterozygote and Zhenshan 97 homozy-gote, respectively. Only four chromosomes contained segments of donor parent, Minghui 63. The other eight chromosomes were W xed with Zhenshan 97Theor Appl Genetwith distance of 16.5cm between plants within a row, and of 25cm between rows on 11 June 2002 in Wuhan (East longitude 114°, North latitude 30°), where the average day length during the period between June and August is 13.5h. BC3F1 ratoons from Hainan were divided into seven individuals and transplanted in the W eld on 1 May 2003. Zhenshan 97, Minghui63, BC2F3-SPP7, the 190 BC2F3 families and the large BC3F2 population with 8,400 plants were sown on 19 May 2003. Plants were then transplanted to the W eld on 10 June 2003 in Wuhan. From each BC2F3 family, 16 healthy plants were selected for the progeny test. Field management essentially followed the normal agricul-tural practices, with fertilizer applied (per hectare) as fol-lows: 48.75kg N, 58.5kg P and 93.75kg K as the basal fertilizer; 86.25kg N at the tilling stage and 27.6kg N at the booting stage. Each plant was harvested individually at maturity.Yield per plant (YD) was scored as the total weight (g) of the grains from the entire plant. The number of tillers per plant (TPP) scored as the number of reproductive tillers for each plant. The number of spikelets per panicle (SPP) was measured as the number of spikelets divided by TPP. Seed setting rate (SR) was scored as the number of grains per panicle divided by SPP, 1000-grain weight (GW) as YD divided by the number of grains multiplied by 1,000. Five plants of Zhenshan 97, Minghui 63, BC2F3-SPP7 and BC3F1 were used to score the phenotype. The arithmetic mean of each homozygous BC2F3 family in the targeted region was regarded as the trait value of the genotype. For the heterozygote family (whose segregation ratio within each family––frequently distorted away from 1:2:1), the weighted mean of the 16 plants was regarded as the trait values of corresponding heterozygous BC2F2 plants. The weighted mean of trait value (y) was calculated by the for-mula: y=a/4+b/4+c/2, in which, a, b and c represent the trait mean value of Zhenshan 97 homozygote, Minghui 63 homozygote and heterozygote genotypes within a family, respectively.Genotyping of populationsGenomic DNA was extracted from 190 BC2F2 plants, 190 BC3F2 plants and 1,082 plants with small panicle (<96 spik-elets) out of 8,400 BC3F2 plants by using simple fast CTAB method (Murray and Thompson 1980). Co-dominant SSR markers located in the region were used to screen the poly-morphism between parents. The SSR genotyping was exe-cuted as described by Wu and Tanksley (1993).Linkage analysis and QTL analysisGenetic linkage map was constructed by using Mapmaker/ EXP 3.0 (Lincoln et al. 1992). The genetic distance was determined by Kosambi function. Due to very few regions segregated in the populations of the 190 BC2F2 plants and 190 BC3F2 plants, simple interval mapping were employed for QTL analyses using Mapmaker/QTL (Lander and Bot-stein 1989). The LOD thresholds ranged from 2.3 to 2.8 for the 5 traits, determined by 1,000 random permutations at a false positive rate of 0.05 for each trait. Performance of the self-pollinated progeny (BC2F3) was used to determine the individual BC2F2 genotypes at the QTL. Then he QTL was treated as a marker and were directly located into the link-age group by using Mapmaker/EXP 3.0.Fine mapping the gene using the extremely small panicle plantsBased upon the assumption that all 1082 small panicle (<96 spikelets) plants were homozygous for the recessive allele (Zhenshan 97) at the targeted locus, the recombination fre-quency between a marker and the gene locus was calculated by using of a maximum likelihood estimator: c=(N1+N2/ 2)/N (Allard 1956; Zhang et al. 1994), in which N is the total number of the extremely small panicle plants investi-gated, N1 is the number of plants homozygous for Minghui 63, and N2 is the number of plants heterozygous for the par-ents. 20 progeny plants of each recombinant were grown in the next normal rice growing season to re-evaluate its phe-notype.ResultsThe trait performance of Zhenshan 97, Minghui 63,BC2F3-SPP7, BC3F1Minghui 63 showed signi W cant higher trait values than Zhenshan 97 in SPP, GW, SR and YD; however, Zhenshan 97 had more TPP (Table1). Except SPP, no signi W cant di V erence was identi W ed in the other four traits between BC3F1 (ratoon) and BC2F3-SPP7 with homozygous Ming-hui 63 alleles in the targeted region. BC2F3-SPP7 and BC3F1 had much higher values in SPP and YD as compared to Minghui 63, the original parent of the population with higher trait values.Phenotypic variations in BC2F2 and BC2F3The frequency distribution of SPP showed discontinuous variation with a clear gap between 110 and 120 spikelets based on the progeny test in the BC2F2 population (Fig.2). According to the progeny test, the BC2F2 individuals could be classi W ed into three subgroups for SPP expressing uni-form large panicle, uniform small panicle and varied SPP among 16 plants within each family. The three subgroupsTheor Appl Genetcorresponded to the three genotypes, respectively: the homozygotes of Minghui 63 (MM) and Zhenshan 97 (ZZ),and heterozygote (MZ) at the targeted QTL. On average,the number of spikelets per panicle of genotype MM was 102 more than that of ZZ. Genotype ZM was intermediate to those of the homozygotes. As expected, the frequency distribution of SPP in the BC 2F 2 population followed theMendelian ratios (1:2:1 by progeny test) for single locus segregation ( 2=1.07, P =0.586). SPP displayed bimodal distribution in the 190 BC 3F 2 (Fig.2). The 190 plants were separated into two distinct groups: plants with small panicle (50 plants) which could be homozygous for Zhenshan 97alleles at the targeted gene locus and plants with large pani-cle (140 plants) which could be the mixture of homozyg-otes for Minghui 63 alleles and heterozygotes. Also,frequency distribution was also in agreement with the expected Mendelian ratio (1:3) for single locus segregation ( 2=0.11, P =0.740). In contrast, the other 4 traits showed continuous but not normal distribution (Fig.2). Therefore,it was impractical to clearly classify them into subgroups according to trait performance by the progeny test.The BC 2F 2 population was classi W ed into three sub-groups of ZZ, MM and MZ according to SPP performance in the progeny test, the average values of the W ve traits were calculated for the three subgroups. Paired t test was used to compare trait value di V erences between subgroups. The averaged values of GW and SR among three subgroupsTable 1Trait performance of Minghui 63, Zhenshan 97, BC 2F 3-SPP7and BC 3F 1 grown in the natural W eld in 2003Values within columns followed by the same letter are not statistically signi W cant (P =0.01) according to Duncan’s test*The trait value of BC 3F 1 was gotten from BC 3F 1 ratoon, which was divided into seven individuals and transplanted in the W eldPlant SPP GW (g)SR (%)TPP YD (g)Zhenshan 97109.2a 23.5a 72.7a 10.5b 19.6a Minghui 63148.6b 28.0b 77.1b 9.4a 29.9c BC 2F 3-SPP7185.4d 22.9a 74.2a 8.8a 27.7b BC 3F 1*165.2c23.8a75.3a9.1a26.9bFig.2Frequency distributions of the traits in BC 2F 2 population and BC 3F 2 population. Black, white and gray bars indicated the genotypes of heterozygous, homozygous for Minghui 63 alleles and homozygousfor Zhenshan 97 alleles in BC 2F 2 population at qSPP7, respectively.The three genotypes at qSPP7 were inferred by BC 2F 3 progeny testTheor Appl Genetindicated no signi W cant di V erence. On the contrary, signi W -cant di V erence existed between ZZ and the other two sub-groups (P =0.001) with regard to YD and TPP. MM genotype had the YD of 26.5g signi W cant higher than that of MZ, 24.2g (P =0.05).The correlation coe Y cients between yield and its four components were presented in Table 2. The correlation coe Y cient between SPP and YD was the highest (r =0.66)among all the investigated pair-tests, signi W cant negative correlations were observed between SPP and SR (r =¡0.29), between SPP and TPP (r =¡0.54). No signi W -cant correlation was detected between SPP and GW (r =0.03).Validation of QTL for SPPIn our previous studies, one putative QTL a V ecting SPP and YD was identi W ed in the pericentromeric region of rice chromosome 7, between RFLP markers C1023 and R1440(Fig.3a). In this study, QTL analysis was further performed in the same target region using BC 2F 2 and BC 3F 2 popula-tions.The genotypes of the corresponding BC 2F 2 plants at qSPP7 locus were easily determined on the performance of their progenies. Thus, qSPP7 treated as a marker waslocated in the locus between SSR marker RM3859 and RFLP marker C39, 0.2cM away from each marker (Fig.3b). qSPP7 showed partial dominance for SPP.In the BC 3F 2 population, one QTL with a LOD value of 56.1 for SPP was located in the interval between RM3859and C39 on chromosome 7. For simplicity, and hereafter,the QTL was referred as qSPP7, which was 0.2cM away from RM3859 and C39, and co-segregated with RM5436.Its additive and dominant e V ects were 59.8 and 28.3 spik-elets per panicle with a contribution of 74.2% to the pheno-typic variance.Accordingly, based on the BC 2F 2 population and its progenies, two major QTLs controlling YD and TPP, were respectively mapped to the same interval where qSPP7 was located (Table 3). Also, a minor QTL for SR was also detected in the same interval. Minghui 63 allele increased SPP and YD, but decreased TPP and SR. No QTL for GW was mapped. The QTL for SR expressed over-dominance,but the others showed partial dominance.High-resolution mapping of qSPP7The 190 BC 3F 2 plants could be divided into two subgroups with 112 spikelets per panicle as the boundary (Fig.2).Zhenshan 97 alleles at qSPP7 exhibited small SPP in con-trast to Minghui 63 alleles and heterozygous alleles. In order to avoid using heterozygous plants, 1,082 plants with extremely small panicle (<96 spiketes), which accounted for about 1/8 of 8,400 BC 3F 2 plants, were selected for map based gene cloning. Two SSR markers of RM5451 and RM445 X anking the QTL (Fig.3) were used to detect the recombinant events between markers and the targeted gene.Analysis of RM5451 identi W ed 66 recombination events between the marker and qSPP7 on one side, and analysis of RM445 detected 146 recombination events between theTable 2Correlation coe Y cient for yield traits calculated with BC 2F 3population *,**Signi W cant at the level of =0.05, 0.01 respectivelyTrait SPP SRYDGWSR ¡0.29**YD 0.66**0.22**GW 0.03¡0.050.20*TPP¡0.54**0.35**¡0.12¡0.03Fig.3Linkage maps showing the location of qSPP7 based on three populations. a Linkage map of chromosome 7 showing the QTL region based on the recombinant inbred lines from the cross between Zhenshan 97 and Minghui 63, the black bar indicated 1-LOD con W dence interval (Xing et al. 2001). b The QTL (qSPP7) location was mapped based on the 190 BC 2F 3 progeny test. The black dot indi-cates the centromere position. c The QTL (qSPP7) location was determined based on 1082 extreme small panicle BC 3F 2 plants. The black dot indicates the centromere positionTheor Appl Genetmarker and qSPP7 on the other side. Assay with four addi-tional markers of RM3859, RM1135, RM7110 and C39,which are more internal to qSPP7, identi W ed 4 recombina-tion events on the RM5451 side and 94, 88 and 4 recombi-nation events on the RM445 side, respectively (Fig.4).Using the formula given by Zhang et al. (1994), the recom-bination frequencies between RM3859 and qSPP7, and between C39 and qSPP7 were both calculated to be 0.18%,which equaled to 0.2cM from qSPP7 to both RM3859 and C39 (Fig.3c). Consequently, two SSR markers RM5436and RM5499 were chosen to screen the eight recombinants from each sides of the gene. However, no recombination event was left for the two markers. RM5436 and RM5499were located in the BAC clones of AP003756 and AP003836, respectively. The physical distance between the two markers is of 912.4kb (/Oryza_sativa_japonica/mapview?chr=7). Thus, the qSPP7was de W ned by RM3859 and C39, and co-segregated with a large region.DiscussionIn this study, a major QTL for SPP was W ne mapped to a 0.4cM region, which had e V ects on TPP, YD and SR as well. This mapping result bene W ted mainly from the use of near isogenic lines and should be of great value for transfer-ring the favorable alleles to rice varieties.The target region exhibited similar e V ects on SPP in both the F 2 population (Yu et al. 1997; 2002) and the RIL popu-lation (Xing et al. 2001; 2002) derived from the same cross between Zhenshan 97 and Minghui 63. The QTL-NIL pop-ulations in the present study revealed even richer informa-tion on this QTL. Besides the same direction of the genetic e V ect with Minghui 63 alleles increasing trait values across di V erent generations, the NIL population provided more accurate estimation of genetic e V ects on SPP due to the minimization of its genetic background noise. Obviously,these genetic e V ects in F 2 and RILs were underestimated.As expected, SPP frequency showed normal distribution in F 2 and RIL populations. In contrast, it expressed a bimodal or discontinuous SPP frequency distribution in the NILs,which could be used to determine the genotype of individ-ual plants at qSPP7 according to their progeny test. In fact,several examples of successful QTL cloning were bene W ted greatly from development of high quality NILs (Yamamoto et al. 2000; Lin et al. 2003) in the last decade. There were few heterozygous plants falling into the category of small number of SPP (Fig.2) in this study could cause false recombinants in the W ne mapping process. This problem was solved in two ways: W rst, only 1/8 of the plants with the smallest number of SPP were chosen from the 8,400 plants to screen the recombinants, which can greatly minimize the risk of false recombinants; second, the reliability of all screened recombinants were further con W rmed by progeny test, which showed uniform small number of SPP among 20 progeny plants. Hence, it is highly con W dent that all recombinants are authentic in this study.Although centromeres are often associated with a depression of meiotic recombination adjacent to theTable 3Genetic e V ects of the QTL region between RM3859 and C39on the four traits by Mapmaker/QTL A additive e V ect on the Minghui 63 allele, D dominant e V ect on the Minghui 63 allele, R 2 percentage of total phenotypic variance explained by the QTL*Genotypes of the corresponding BC 2F 2 plants at the gene locus were identi W ed by BC 2F 3 progeny test, and then the QTL treated as a single Mendelian factor was mapped to the linkage group. Its genetic e V ect estimated on the progeny data. Additive e V ects equal to the half of the trait value di V erence between two homozygotes, dominance e V ects equal to the trait value di V erence between heterozygote and the middle value of two homozygotesTraits Population LODA D D /A R 2 (%)SPP BC 2F 3*51.124.70.48BC 3F 256.159.828.30.4774.2YD (g)BC 2F 322.3 4.4 2.70.6143.2TPP BC 2F 332.4¡1.6¡1.00.6361.9SR (%)BC 2F 33.0¡1.6¡2.11.317.4Fig.4Genetic and physical map in the local region of qSPP7.Numbers of recombinants detected in 190 BC 2F 2 plants and 1082BC 3F 2 plants between the markers and qSPP7 were indicated in the parentheses in the top and bottom of markers, respectively. SSR mark-er physical position was given at two forms of physical distance andthe accession number of BAC clone contained the SSR marker (/Oryza_sativa_japonica/). The genetic positions of SSR markers were mined from the website http://rgp.dna.affrc.go.jp/cgi-bin/statusdb/stattable.pl?chr=7&lab=RGPTheor Appl Genetpericentromeric regions (Haupt et al. 2001), no recombina-tion suppression was reported in the pericentromeric region on rice chromosome 3 (Fan et al. 2006). Thus, it is possible to narrow down the QTL located in the pericentromeric regions to a candidate gene by map-based cloning in this study. Unfortunately, severe recombination suppression was observed in the targeted interval within pericentro-meric region (Fig.4). The recombinants between markers RM5451, RM445 and RM7110, and qSPP7 in the 1082 BC3F2 plants were about W ve-fold as those in the 190 BC2F2 plants, respectively. While, the recombinants in the 1082 BC3F2 plants decreased to four-fold as those in the 190 BC2F2 plants for markers RM3859 and C39, indicating the presence of light recombination suppression at the two marker loci. There was no recombinant in both two popula-tions for markers RM5436 and RM5499 covered a distance of more than 900kb. This indicated the presence of severe recombination suppression in the region between RM5436 and RM5499. No clear recombination suppression was observed in the regions between RM5451 and RM3859, RM1135 and RM7110. But clear recombination suppres-sion was occurred in the region between RM7110 and C39. Therefore, recombination suppression was occurred at least in the region between RM3859 and C39 although the right boarder of recombination suppression region could not be precisely de W ned due to lack of polymorphic markers in the large region between RM7110 and C39. The genetic dis-tance between RM3859 and C39 was 0.4cM based on mapping result in either 190 BC3F2 plants or 1082 extreme BC3F2 plants. RM3859 and C39 are located in the BAC clones AP004176 and AP004346, respectively. The physi-cal distance between these two clones was estimated to be as large as 2,400kb or so (/Oryza_ sativa_japonica/mapview?chr=7). The ratio of physical-to-genetic distance in this targeted region was about 6,000kb/ cM, which is 20 times higher than the estimated average ratio (250–300kb/cM) (Arumuganathan and Earle 1991). This is consistent with the ratio of 2740kb/cM, or 10 times higher than that of the rest of the genome reported by Wu et al. (2002). In tomato, it can be as high as 10- to 40-fold (Tanksley et al. 1992). In the present study, no recombi-nant was found between qSPP7 and markers in the large region of 912.4kb. Therefore, we failed to further narrow down the gene using a large population consisting of 1082 plants after qSPP7 was mapped to a 0.4-cM region using the small BC2F2 population consisted of 190 individuals. Taken together, there was no resolution di V erence between the large and small populations for QTL W ne mapping. That is to say, resolution of gene mapping could not be improved for a region with clear recombination suppression by enlarging a population even though there are lots of polymorphic markers in the region. This comes up with the conclusion that positional cloning strategy would not be feasible for isolation of the targeted gene, qSPP7.Besides GW, one QTL for YD and TPP and SR was simultaneously detected in the qSPP7 surrounding region. The QTL for SR expressed over-dominance, the QTL for other traits acted in partially dominant (Table3). These indicated that the homozygote with Minghui 63 alleles is promising to produce higher yield than does the heterozy-gote. Given the traits of yield components are highly corre-lated, ridge regression analysis was used for the phenotype data set collected from BC2F3 to build a regression equation of the form: YD=¡70.454+0.16£SPP+2.08£TPP+ 0.92£GW+39.23£SR. From the equation, the theoreti-cal YD can be easily predicted. For example, the additive and dominance e V ects of qSPP7 were estimated in BC2F3, the theoretical YD di V erence between genotypes MM and MZ at qSPP7 is 3.2g. This is well illustrated by the fact that the Minghui 63 homozygotes at qSSP7 showed larger average values in the traits than the heterozygotes in the BC2F3 progenies. Moreover, the averaged grain yield per plant of Minghui 63 homozygotes was 26.5g, which was signi W cantly higher (P=0.05) than heterozygotes of 24.2g in the BC2F3 progenies. This result was also consistent with the conclusion that heterozygotes were not always advanta-geous for F1 performance on the basis of an “immortalized F2” population produced from the same cross between Zhenshan 97 and Minghui 63 (Hua et al. 2002). Thus, the ideal genotype in the qSPP7 surrounding region in the hybrid should be Minghui 63 homozygotes for hybrid pro-duction. Markers X anking the QTL could be directly used for improvement of parent Zhenshan 97. Theoretically, the new hybrid Shanyou 63 with homozygous Minghui 63 alle-les at qSPP7 should exhibit higher YD than does its origi-nal hybrid Shanyou 63, an elite rice hybrid between Zhenshan 97 and Minghui 63 in last two decades in China. The new version Shanyou 63 is in the process of develop-ment.In summary, the QTL of qSPP7 will play a crucial role in developing high yielding rice varieties. E V orts in its iso-lation are worth being made in combination with bioinfor-matic strategy.Acknowledgments The authors thank Dr. Cheng JH for helpful lan-guage improvement. This work was supported by a grant from the Na-tional Natural Science Foundation of China (30470987), and a grant from the National Key Program on Basic Research and Development of China.ReferencesAllard RW (1956) Formulas and tables to facilitate the calculation of recombination values in heredity. Hilgardia 24:235–278 Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Mol Biol Rep 9:211–215。

增加穗粒数的水稻染色体代换系Z747鉴定及相关性状QTL定位王大川; 赵芳明; 汪会; 马福盈; 杜婕; 张佳宇; 徐光益; 何光华; 李云峰; 凌英华【期刊名称】《《作物学报》》【年(卷),期】2020(046)001【总页数】7页(P140-146)【关键词】水稻; 染色体片段代换系; 粒数; QTL定位【作者】王大川; 赵芳明; 汪会; 马福盈; 杜婕; 张佳宇; 徐光益; 何光华; 李云峰; 凌英华【作者单位】西南大学水稻研究所/西南大学农业科学研究院重庆 400715【正文语种】中文水稻是世界上最重要的粮食作物之一[1], 提高水稻产量以保障世界粮食安全刻不容缓。

水稻产量主要由穗数、粒数、粒重和结实率构成。

每穗粒数由穗长、一次枝梗和二次枝梗数决定[2], 所以增加每穗粒数是提高水稻产量的途径之一。

然而粒数属于数量性状, 由多基因控制。

水稻染色体片段代换系可将多位点控制的复杂性状分解, 使QTL定位更加准确, 尤其定位出的QTL可直接应用于育种实践, 因而是理想的遗传材料。

到目前为止, 已经克隆了许多与水稻粒数相关的基因。

其中一些涉及细胞分裂素、生长素和茉莉酸等激素信号途径。

如Gn1a (Grain number 1a) [3]表达量降低引起花序分生组织中细胞分裂素的积累, 从而增加水稻粒数。

GNP1 (Grain Number per Panicle 1) [4]通过增加水稻穗分生组织中的细胞分裂素活性,提高籽粒数目和产量。

An-1 (Awn-1)[5]基因表达上调会引起一个重要的细胞分裂素调控基因LOG的表达下调, 使分生组织的活性降低, 减少每穗粒数。

OsGRF6[6]能与OsTAWAWA1及OsMADS34启动子结合, 正调控生长素的生物合成和信号转导, 促进花序发育, 增加穗粒数。

PAY1 (Plant Architecture and Yield 1)[7]通过影响生长素极性运输和改变内源吲哚-3-乙酸的分布改善水稻株型, 进而增加水稻穗粒数。

研究生课程论文课程名称作物遗传与分子育种开课时间2013-2014学年第一学期学院化学与生命科学学院学科专业遗传学学号**********姓名蒋续续学位类别全日制硕士任课教师马伯军交稿日期成绩评阅日期评阅教师签名植物细胞分裂素及其研究进展蒋续续（浙江师范大学化学与生命科学学院，浙江金华321004）摘要：细胞分裂素是一类重要的植物激素，在植物的生长发育过程中起着重要的作用。

随着近年来深入的研究，其化学结构与生理功能已经研究地十分透彻。

而且细胞分裂素与其他激素相互作用调控植物发育的相关研究也已经展开并取得了可喜的进展。

由于它的作用机理已经被人们研究清楚，所以在农业上的到了广泛的应用，提高了作物的产量与品质，大大促进了农业的发展。

关键词：细胞分裂素，生理结构，应用Plant Cytokinin and Its Research Progress Abstract：Cytokinin is a kind of important plant hormone and plays an important role in the process of plant growth. With in-depth research in recent years, its chemical structure and physiological function has been studied very well. And the study of interaction between cytokinin and other hormones in regulating plant development has begun and got the gratifying progress. Because of its mechanism of action has been clear, so it has widespread application in agriculture, increasing the crop yield and quality, greatly promoting the development of agriculture. Keywords：cytokinin，Physiological structure，application1 细胞分裂素发现历史斯库格和崔澄等在寻找促进组织培养中细胞分裂的物质时，发现生长素存在时腺嘌呤具有促进细胞分裂的活性。

Journal of Integrative Plant Biology 2006, 48 (11): 1300−1305Received 28 Mar. 2006 Accepted 24 Apr. 2006Supported by the State Key Basic Research and Development Plan of China (G1999011603–2005CB120802) and the National Natural Science Foundation of China (30570969).*Author for correspondence. Tel: +86 (0)20 8528 1908; Fax: +86 (0)20 85280200; E-mail: <ygliu@>.© 2006 Institute of Botany, the Chinese Academy of SciencesA Rice Panicle Mutant Created by Transformationwith an Antisense cDNA LibraryYuan-Ling Chen, Qun-Yu Zhang, Yu-Yu Jian, Yue-Sheng Yang,Kai-Dong Liu and Yao-Guang Liu *(Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong, College of Life Sciences ,South China Agricultural University , Guangzhou 510642, China)AbstractA rice (Oryza sativa L.) mutant displaying defects in panicle development was identified among transformants in a transgenic mutagenized experiment using an antisense cDNA library prepared from young rice panicles.In the mutant, the average spikelet number was reduced to 59.8 compared with 104.3 in wild-type plants. In addition, the seed-setting rate of the mutant was low (39.3%) owing to abnormal female development.Genetic analysis of T 1 and T 2 progeny showed that the traits segregated in a 3 (mutant) : 1 (wild type) ratio and the mutation was cosegregated with the transgene. Southern blot and thermal asymmetric interlaced polymerase chain reaction analyses showed that the mutant had a single T-DNA insertion on chromosome 5, where no gene was tagged. Sequencing analysis found that the transgenic antisense cDNA was derived from a gene encoding an F-box protein in chromosome 7 with unidentified function. This and another four homologous genes encoding putative F-box proteins form a gene cluster. These results indicate that the phenotypic mutations were most likely due to the silencing effect of the expressed transgenic antisense construct on the member(s) of the F-box gene cluster.Key words: antisense gene silencing; F-box protein; panicle development; rice.Chen YL, Zhang QY, Jian YY, Yang YS, Liu KD, Liu YG (2006). A rice panicle mutant created by transformation with an antisense cDNA library. J Integr Plant Biol 48(11), 1300−1305.doi: 10.1111/j.1672-9072.2006.00355.x; available online at , Identification of loci that control agronomic traits and under-standing their molecular functions are important for both basic biology and crop breeding. Grain number and seed-setting rate are important traits that directly contribute to yield in rice production. The former is affected by several factors, such as branch number, total branch length, and spikelet density,whereas the latter depends on the development of male and female organs and gamete fertility. However, little is known about the molecular network controlling rice reproductivedevelopment. It is known that rice grain number per panicle is controlled by a number of quantitative trait loci. One of these,Gn1a encoding a cytokinin oxidase/dehydrogenase, has been isolated recently and subsequently named OsCKX2 (Ashikari et al. 2005). Reduced expression of OsCKX2 leads to enhanced accumulation of cytokinin in inflorescence meristems and re-sults in increases in grain number per panicle.T-DNA insertional mutagenesis and transposon tagging are useful tools to create mutants for gene isolation and functional studies in plants (Walbot 1992). There have been other ap-proaches derived that are similarly useful and, among them, is the random introduction of an antisense transgene into the plant genome and silence the target gene by using an antisense cDNA library. We have created a rice mutagenized library by rice transformation with an antisense cDNA library (Chen et al.2004). Herein, we report a rice panicle mutant obtained from this mutant library that exhibited decreases in both spikelet number and seed-setting rate. The results demonstrate that theA Rice Panicle Mutant Created by Antisense Mutagenesis 1301mutations were possibly caused by expression of a transgenic antisense cDNA derived from a member of an F-box gene cluster, suggesting a possible role for F-box proteins in rice reproductive development.ResultsA transgenic mutant displayed reductions in both spike-let number and female fertilityA mutant was obtained from transformation of the japonica variety Zhonghua 11 with an antisense cDNA library prepared from young rice panicle RNA (Chen et al. 2004). The mutant showed reductions in both spikelet number and seed-setting rate (Figure 1). The average spikelet number and seed-setting rate in T 1 and T 2 plants were 59.8% and 39.3%, respectively,compared with 104.3% and 90.3% in the wild type. As a result,the mutant had only 25.0% of filled grain per panicle compared with the wild type.Although the length of the panicle (approximately 20–21 cm)in the mutant was similar to that of wild type (Figure 1), the number of primary and secondary branches, spikelet number per branch, and spikelet density (number of spikelets/cmbranch) in the mutant were significantly lower than in the wild type (Figure 2). The number of primary and secondary branches in the mutant was 6.4 and 17.3, respectively, with 2.5 and 7.9less than in the wild type, respectively (Figure 2A). Spikelet numbers per primary and secondary branches in the mutant were 10.6 and 2.3, respectively, which were 3.5 and 0.7 less than those of the wild type, respectively (Figure 2B). The num-ber of spikelets/cm branch in the mutant and wild type was 0.90 and 1.24, respectively (Figure 2C).The pollens of the mutant, examined by staining with potas-sium iodide, were morphologically normal (data not shown).We further examined the germination ability of the pollen in a medium, as described previously (Wang et al. 2000). It was found that the germination rate (7.8%) of pollen in the mutant was approximately the same as that of the wild type (7.3%),confirming that male fertility in the mutant was normal. Moreover,a saturated pollination experiment was performed in which a large amount of fertile pollen from wild-type plants was applied to the flowering spikelets of the mutant. The seed-setting rate observed in this experiment was 32.5%, similar to that of self-pollinated mutant panicles. These results indicate that the de-creased seed-setting rate in the mutant could be attributed to abnormal female development.Mutant phenotypes were cosegregated with the transgenic eventA total of 131 T 1 plants was investigated for segregations of the traits and the T-DNA insertion. It was found that 92 and 39plants exhibited mutated and wild-type phenotypes,respectively, fitting the predicted 3 : 1 segregation ratio (χ2 =1.35<3.84=χ20.05) of single-locus inheritance (Table 1). Analy-sis of these T 1 plants showed that all plants with the mutant phenotype carried the T-DNA and were hygromycin resistant,whereas the wild-type plants lacked the T-DNA and werehygromycin sensitive. Thus, the mutant phenotypes wereFigure 1. Panicles of (A) mutant and (B) wild-type T 1 plants with and without the T-DNA, respectively.Figure 2.Characterization of the panicle of the mutant and wild-type plants.1302 Journal of Integrative Plant Biology Vol. 48 No. 11 2006cosegregated with the T-DNA insertion and were inherited dominantly.To test whether the mutant phenotypes were inherited stably,we further examined the T 2 progeny. Among the 13 T 2 lines examined, two were derived from wild-type T 1 plants that con-tained no insertion of the T-DNA and three and eight were from the progeny of plants homozygous and heterozygous for the insertion, respectively (Table 2). In the heterozygous lines, the abnormal spikelet number and seed-setting rate were again cosegregated with the T-DNA (Hm resistance) and the pheno-types of the homozygous wild-type and mutant lines were com-pletely correlated with the absence or presence of the T-DNA.The mutant carried a single T-DNA insertionSouthern blotting analysis was performed to examine the num-ber of T-DNA insertions in the mutant. When genomic DNA was digested with Hin dIII, which is a unique site within the T-DNA region (Figure 3A), only one band was detected (Figure 3B),indicating that the mutant was derived from a single T-DNA insertion. We then amplified the genomic sequence flanking the T-DNA insertion using thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) (Liu et al. 1995, 2005) (Figure 3C).Sequencing of the TAIL-PCR product and BLAST analysisshowed that the T-DNA was integrated into a site on the chro-mosome 5, around which there was no coding sequence, sug-gesting that the mutant was not caused by a T-DNA tagging in a functional gene.The transgenic antisense cDNA in the mutant was de-rived from an F-box geneThe transgenic antisense cDNA (1 051 bp) was amplified from the mutant DNA by PCR using primers flanking the cloning site in the T-DNA construct (Figure 4A). Reverse transcription (RT)-PCR analysis confirmed that the chimeric anti-cDNA/mgfp con-struct driven by the CaMV35S promoter (Figure 3A) was ex-pressed in the mutant (Figure 4B). Sequencing analysis showed that the original cDNA was derived from a predicted gene on the rice chromosome 7, which is located in the 82.3–88.5 kb region of the genomic clone P0554D11 (Figure 4C). This gene was predicted to encode a putative F-box protein. Further se-quence analysis indicated that this gene and four other ho-mologous genes, all encoding proteins with a conserved F-box domain, form a gene cluster within an approximate 35-kb region (Figure 4C). The antisense cDNA fragment is comple-mentary to all these genes in various lengths, ranging from 1 051 to 100 bp.Table 1. Segregation of the phenotypic mutations and T-DNA /hygromycin resistance in T 1 progenyPhenotype No. plantsNo. spikelets Seed-setting rate (%)T-DNA/Hm test Mutant 9255.1 ± 1.242.2 ± 1.5Present/resistant Wild type3996.6 ± 2.292.0 ± 0.7Absent/sensitiveData are the mean ± SE . Hm, hygromycin.Table 2. Segregation of the phenotypic mutations in T 2 progenyLinesNo. spikelets Seed-setting rate (%)Hm-R Hm-S Hm-R Hm-S ZE9-34115.6 ± 5.785.7 ± 2.3ZE9-36123.5 ± 2.382.9 ± 1.3ZE9-1258.0 ± 1.230.6 ± 1.6ZE9-3251.3 ± 1.129.5 ± 1.5ZE9-3562. 7± 2.342.7 ± 3.1ZE9-1672.0 ± 3.6120.4 ± 6.230.2 ± 4.882.5 ± 3.6ZE9-2461.4 ± 2.3101.2 ± 4.927.5 ± 1.882.6 ± 8.8ZE9-2579.9 ± 4.2126.9 ± 6.540.8 ± 4.193.3 ± 1.6ZE9-3166.3 ± 1.8106.1 ± 11.030.7 ± 2.278.4 ± 7.1ZE9-4282.1 ± 2.3129.7 ± 4.043.6 ± 3.484.7 ± 2.2ZE9-4496.3 ± 1.5137.0 ± 2.831.1 ± 1.288.2 ± 1.0ZE9-4692.6 ± 1.5120.2 ± 4.330.0 ± 2.486.8 ± 4.4ZE9-5559.6 ± 1.690.4 ± 7.530.7 ± 2.186.9 ± 6.5Average64.4111.936.488.6Data are the mean ± SE . R, resistant; S, sensitive.A Rice Panicle Mutant Created by Antisense Mutagenesis 1303DiscussionIn the present study we characterized a panicle mutant and found that the reduced spikelet number was due to decreases in the number of primary and secondary branches, as well as a reduction in spikelet density on the branches, whereas the low seed-setting rate was attributed to abnormal female development. Segregation analysis demonstrated that the two mutated traits were correlated with a single transgenic event.Because the mutant was generated from a rice mutant library by transformation with an antisense cDNA library (Chen et al.2004), there are two possible causes for the mutation: (i) inac-tivation or tagging of a functional gene by the T-DNA insertion;or (ii) the silencing of endogenous gene(s) by the transgenicantisense RNA. The results showed that the mutations were inherited in a dominant manner (i.e. heterozygous transgenic plants exhibited the mutant phenotype and their progeny showed a 3 (mutant) : 1 (wild type) segregation ratio). This is consis-tent with the notion that the mutation is caused by antisense gene silencing. Furthermore, analysis of the T-DNA insertion site showed no endogenous gene to be destroyed. These data strongly suggest that the mutation should be generated by antisense mutagenesis.The F-box proteins belong to a large gene family in plants (Goff et al . 2002; Risseeuw et al. 2003). However, only a few F-box proteins have been functionally studied in plants and, in rice, only one case has been reported (Ishikawa et al. 2005). It is known that F-box proteins play an essential role in the SCF (Skp-Cullin-F-box) complex in recruiting protein substrates for ubiquitin-dependent protein degradation; specific proteolysis in plants plays an important role in regulating plant growth and development (Xiao and Jang 2000; Risseeuw et al. 2003). It has been reported that many crucial biological processes in plants are governed by a ubiquitin/26S proteosome-mediated specific protein degradation pathway in which F-box proteins are involved. These include embryogenesis and senescence(Moon et al. 2004), flower development (Wang et al. 2003),Figure 3. Characterization of the T-DNA insertion in the mutant.(A) Structure of the T-DNA region for the transformation, which was derived from the binary vector pCAMBIA1302. The antisense cDNA fragment is driven by the CaMV35S promoter (P 35S ). Small arrows indicate primers for polymerase chain reaction (PCR) amplification of the antisense cDNA fragment (P1, P2) and reverse transcription-PCR test of the expression of the construct (P3, P4).(B) Southern blot hybridization with the hpt (hygromycin phosphotransferase) gene as a probe. Wild-type and mutant ge-nomic DNAs were digested with Hin dIII and the binary plasmid containing the T-DNA structure was cut with Xho I.(C) Amplification of the mutant genomic sequence flanking the T-DNA insertion by thermal asymmetric interlaced PCR. II and III indicate secondary and tertiary reactions for TAIL-PCR, respectively.Figure 4. Analyses of the antisense cDNA construct.(A) The polymerase chain reaction (PCR) amplification of the antisense cDNA in the mutant using the primers P1 and P2 (see Figure 3A). Lane 1, mutant DNA; lane 2, wild-type DNA.(B) The reverse transcription-PCR test of the construct in the mutant using the primers P3 and P4. Lane 1, mutant genomic DNA; lane 2,mutant RNA without reverse transcription; lanes 3, 4, mutant RNA with reverse transcription.(C) Location of the antisense cDNA on a putative F-box gene cluster on chromosome 7 corresponding to a genomic clone P0554D11(accession no. AP004569). The arrow indicates the antisense cDNAfragment. Predicted introns in these genes, if any, are not shown.1304 Journal of Integrative Plant Biology Vol. 48 No. 11 2006photomorphogenesis and flowering time (Somers et al. 2004), self-incompatibility (Qiao et al. 2004), hormone (gibberellic acid, jasmonic acid, and ethylene) responses (Xu et al. 2002; Guo and Ecker 2003; Itoh et al. 2003; Potuschak et al. 2003; Sasaki et al. 2003), and rice tillering (Ishikawa et al. 2005).The results of the present study suggest that F-box proteins may play important roles in the development of the panicle and female organ (or female gametes). Considering that the antisense RNA is complementary to members of the F-box gene cluster, it is possible that the phenotypic mutations were caused by antisense silencing of different genes. Detailed analyses using gene-specific fragments for antisense gene silencing and/or RNA interference are necessary to link the phenotypic mutations with the molecular functions of the members in this gene cluster. Understanding the molecular mechanism control-ling rice reproductive development will pave the way to rice improvement by molecular breeding.Materials and MethodsPlant materials and phenotype investigationThe panicle mutant was screened from a mutant library cre-ated by transforming the japonica rice variety Zhonghua 11 with an antisense cDNA library of young panicle RNA (Chen et al. 2004). An Agrobacterium-mediated method was used for rice transformation and the hygromycin phosphotransferase (hpt) gene was used as a plant selection marker.For investigations of the number of branches per panicle and spikelets per branch in the mutant and wild type (Figure 2), the sample sizes were as follows: for branches per panicle, n=85 and 89 panicles for wild type and mutant, respectively; for spikelets per branch, n=754 and 572 primary branches for wild type and mutant, respectively, and 2 138 and 1 535 secondary branches for wild type and mutant, respectively. The wild-type plants used as a control were selected from T2 and T2 progeny carrying no T-DNA.Hygromycin selection and PCR analysis of the hpt gene in transgenic plants were performed at the tillering stage. The Hm-resistance assay was performed according to the meth-ods reported by Liu et al. (2001). Three pieces of fresh leaves from each plant were cut into 2 cm lengths and immersed imme-diately into a selection solution with 50 mg/L Hm and 1 mg/L 6-benzylaminopurine for 5–7 d at room temperature with a natural photoperiod. The presence of the T-DNA was confirmed by PCR with hpt-specific primers (5'-GTCCGTCAGGACATTGTTGGAG-3'/5'-GTCTCCGACCTGATGCAGCTCTCGG-3').Sequence analysisThe genomic sequence flanking the T-DNA insertion was obtained by TAIL-PCR (Liu et al. 1995, 2005). The specific prim-ers for primary, secondary, and tertiary reactions respectively were as follows: 5'-TGCATGACGTTATTTATGAGATGGGTT-3', 5'-TATGATTAGAGTCCCGCAATTATACA-3', and 5'-ATTATCGCGCGCGGTGTCA-3', respectively. The arbitrary de-generate primer was 5'-NGTCGA(G/C)(A/T)GANA(A/T)GTT-3' (where N represents A, T, C, or G). The amplified fragment from the tertiary reaction was recovered, cloned, and sequenced. The antisense cDNA fragment in the transgenic T-DNA of the mutant was amplified by PCR using two specific primers, n amely 5'-CACGGGGGACTCTTGACCATGG-3' (P1) a nd 5'-TTTGTGCCCATTAACATCACCATCT-3' (P2). The sequences were analyzed by BLAST (/) and the RiceGAAS system (http://rgp.dna.affrc.go.jp). The primers for the RT-PCR test of the expression of the anti-cDNA/mgfp construct were 5'-CGTCAACAGGATCGAGCTTAAG-3' (P3) and 5'-GGTGGTGGCTAGCTTTGTATAG-3' (P4).ReferencesAshikari M, Sakakibara H, Lin S, Yamamoto T, Takashi T, NishimuraA et al. (2005). Cytokinin oxidase regulates rice grain production.Science309, 741–745.Chen YL, Zhang QY, Jian YY, Yang YS, Liu YG (2004). Generation of a rice mutant library by shotgun antisense gene silencing and mutant screening. J South China Agr Univ25, 53–57.Goff SA, Ricke D, Lan TH, Presting G, Wang R, Dunn M et al.(2002). A draft sequence of the rice genome (Oryza sativa L. ssp.japonica). Science296, 92–100.Guo H, Ecker JR (2003). 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GNA11、GNA14在内皮细胞模型中的表达及其功能学研究的开题报告一、研究背景与意义G蛋白（G-protein）是细胞中信号转导的重要分子，其作用是调节细胞内的代谢、生长和分化等生理功能。

G蛋白可以分为Gs、Gi、Gq、G12/13等多个家族，其各自通过与不同的效应蛋白结合从而调节不同的信号通路。

其中，Gq家族成员GNA11和GNA14在内皮细胞中起着重要的作用，能够介导刺激性荷尔蒙和生长因子等信号物质的信号转导，进而调节血管紧张性、血管内皮细胞增殖、炎症反应等多种生物学功能。

因此，研究GNA11和GNA14在内皮细胞中的表达及其功能对于深入了解内皮细胞的生理功能、阐明其参与的信号通路及其在疾病中的作用机制具有重要意义。

另外，此类研究还能为相关药物的开发提供有效的靶点和策略。

二、研究内容和方法1. 研究内容本研究计划通过以下两个方面来深入研究GNA11和GNA14在内皮细胞中的表达及其功能：（1）检测不同来源的内皮细胞中GNA11和GNA14的表达情况，采用Western blot和实时荧光定量PCR技术检测内皮细胞中GNA11和GNA14的表达水平，比较不同来源内皮细胞中GNA11和GNA14的表达差异。

（2）研究GNA11和GNA14在内皮细胞中的生物学功能，通过过表达和下调的方式，比较其对内皮细胞增殖、迁移、血管紧张性和炎症反应等生物学功能的影响。

2. 研究方法（1）内皮细胞的分离和培养：本研究将采用人类动脉内皮细胞（HAECs）、人类肺动脉内皮细胞（HPAECs）和人类脐静脉内皮细胞（HUVECs）等来源的内皮细胞，采用相应的方法分离和培养。

（2）蛋白质和RNA的提取和检测：本研究将采用Western blot和实时荧光定量PCR技术检测GNA11和GNA14在内皮细胞中的表达水平。

（3）细胞增殖和迁移实验：通过MTT检测和划痕实验比较GNA11和GNA14对内皮细胞增殖和迁移的影响。

植物生理学词汇植物生理学 plant physiology message transportation 信息传递signal transduction 信号转导water potential 水势solute potential 溶质势osmotic potential 渗透势matric potential 衬质势diffusion 扩散osmosis 渗透plasmolysis 质壁分离deplasmolysis 质壁分离复原pressure potential 压力势turgor pressure 膨压bulk flow 集流aquaporin 水孔蛋白active absorption of water 主动吸水root pressure 根压blooding 伤流blooding sap 伤流液guttation 吐水apoplast 质外体symplast 共质体passive absorption of water 被动吸水transpiration pull 蒸腾拉力bound water 束缚水gravitational water 重力水cappilary water 毛细管水permanent wilting coefficient 永久萎蔫系数accumulation 积累性lenticular transpiration 皮孔蒸腾cuticular transpiration 角质层蒸腾stomatal transpiration 气孔蒸腾transpiration rate 蒸腾速率transpiration ratio 蒸腾效率transpiration coefficient 蒸腾系数small pore diffusion law 小孔扩散律feed back manner 反馈调节feed forward manner 前馈调节vapor pressure difficiency 叶片-大气水气压亏缺cohesion theory 内聚力学说mineral nutrition 矿质营养ash 灰分ash element 灰分元素mineral element 矿质元素essential element 必需元素solution culture method 溶液培养法water culture method 水培法sand culture method 沙基培养法balance solution 平衡溶液major element 大量元素minor element 微量元素nutrient deficiency symptom 营养缺乏症calcium binding proteins 钙结合蛋白calmodulin 钙调素beneficial elements 有益元素rare earth element 稀土元素selective absorption 选择吸收physiologically acid salt 生理酸性盐physiologically alkaline salt 生理碱性盐physiologically neutral salt 生理中性盐toxicity of single salt 单盐毒害ino antagonism 离子拮抗ion transporter 离子运载体ion channel 离子通道ion carrier 离子载体ion pump 离子泵voltage sensor 电压感受器permease 透过酶transport enzyme 运输酶ATPase ATP酶electrogenic pump 致电离子泵electroneutral pump 中性离子泵masterenzyme 主宰酶pinocytosh 胞饮作用simple diffusion 单纯扩散facilitated diffusion 协助扩散primary active transport 初始主动运输secondary active transport 次级主动运输co-transport 协同转运，共转运secondary co-transport 次级共转运proton mative force 跨膜质子电动势symport 同向转运antiport 反向转运ATP-binding complex ATP结合复合体contact exchange 接触交换foliar nutrition 叶片营养ectodesmata 外连丝nitrate reductase ，NR 硝酸还原酶induced enzyme 诱导酶adaptive enzyme 适应酶nitrite reductase 亚硝酸还原酶glutamine synthetase 谷氨酰胺合成酶glutamate synthase 谷氨酸合成酶carbon assimilation 碳素同化作用photosynthesis 光合作用chlorophyll 叶绿素phytol 植醇carotenoid 类胡萝卜素carotene 胡萝卜素lutein 叶黄素pigment protein complex 色素蛋白复合体phycobillin 藻胆素photon 光子quantum 光量子ground state 基态excited state 激发态light reaction 光反应dark reaction 暗反应reaction centre pigments 反应中心色素light-harvesting pigments 聚光色素antenna pigments 天线色素reaction center 反应中心photosynthetic unit 光合单位exciton transfer 激子传递resonance transfer 共振传递primary electron donor 原初电子供体primary electron acceptor 原初电子受体secondary electron donor 次级电子供体trap 陷阱red drop 红降现象Emerson effect 爱默生效应PSII light harvesting complex，LHC II : PSII 的捕光色素复合体pheo 去镁叶绿素plastoquinone 质体醌reductive pentose phosphate pathway，RPPP 还原戊糖磷酸途径GAP 甘油醛-3-磷酸z scheme z方案cytochrome，Cyt 细胞色素ferrdoxin,Fd 铁氧还蛋白plastocyanin,PC 质蓝素water photolysis 水的光解Hill reaction 希尔反应oxygen-evolving complex,OEC 放氧复合体manganese stablizing protein 锰稳定蛋白water oxidizing clock 水氧化钟noncyclic electron transport 非环式电子传递cyclic electron transport 环式电子传递pseudocyclic electron transport 假环式电子传递water-water cycle 水-水循环photosynthetic phosphorylation 光合磷酸化DNP 二硝基苯酚（解偶联剂）crassulacean acid metabolism,CAM 景天酸代谢the Calvin cycle 卡尔文循环oxaloacetic acid 草酰乙酸malic acid,Mal 苹果酸aspartic acid ,Asp天冬氨酸bundle sheath cell 维管束鞘细胞pyruvic acid 丙酮酸photo respiration 光呼吸light compensation point 光补偿点light saturation 光饱和现象photoinhibition of photosynthesis 光抑制midday depression 午睡现象aerobic respiration 有氧呼吸anaerobic respiration 无氧呼吸respiratory substrate 呼吸底物fermentation 发酵respiratory rate 呼吸速率respiratory quotient 呼吸商glycolysis 糖酵解tricarboxylic acid cycle 三羧酸循环elicitor 激发子biological oxidation 生物氧化barbital acid 巴比妥酸rotenone 鱼藤酮positive effector 正效应物negative effector 负效应物Pasteur effect 巴斯德效应maintenance respiration 维持呼吸growth respiration 生长呼吸confocal laser canning microscope 共聚集激光扫描显微镜empty seed coat technique 空种皮杯技术symplastic phloem loading 共质体装载apoplasmic phloem loading 质外体装载first messenger 第一信使chemical signal 化学信号positive chemical signal 正化学信号negative chemical signal 负化学信号physiocal signal 物理信号action potentials 动作电位variation potentials 变异电位receptor 受体blue light recepter 隐花色素，蓝光受体protein kinase 蛋白激酶phosphorlation 磷酸化作用transcription factor 转录因子plant growth substance 植物生长物质phytohormones 植物激素plant growth regulators 植物生长调节剂auxin 生长素IAA 吲哚乙酸hormone receptor 激素受体acid-growth theory 酸生长学说Gibberellins 赤霉素Gibberellic acid,GA 赤霉酸zeatin 玉米素cytokinin，CTK 细胞分裂素cytokinin oxidase,CKO 细胞分裂素氧化酶abscisin II 脱落素IIdormin 休眠素abscisic acid,ABA 脱落酸terpenoid pathway 类萜途径carotenoid pathway 类胡萝卜素途径xanthoxin 黄质醛lutein 叶黄素neoxanthix 新黄质brassinolide 油菜素内酯brassinosteroide 油菜素甾体类化合物bean bioassay 生物鉴定法jasmonates 茉莉酸Jasmonic acid methyl ester 茉莉酸甲酯linolenic acid α-亚麻酸cyclic fatty acid 环脂肪酸aspirin 阿司匹林salicylic acid ,SA 水杨酸polyamines,PA 多胺putrescine，Put 腐胺cadaverine,Cad 尸胺spermidine,Spd 亚精胺spermine,Spm 精胺CEPA 乙烯利NAA 萘乙酸NAD 萘乙酰胺6-BA 6-苄基腺嘌呤CCC 矮壮素PP333 多效唑growth 生长differentiation 分化development 发育chemical creep 化学滑行totipotency 细胞全能性polarity 极性explant 外植体tissue culture 组织培养primary culture 初代培养subculture 继代培养redifferentiation 再分化embryoid 胚状体somatic embryo 体细胞胚adventitious embryo 不定胚virus-free plants 无病毒植株light seed 需光种子dark seed 需暗种子seed longevity 种子寿命seed vigor 种子活力seed viability 种子生活力seed aging 种子老化determinate 有限性meristem 分生组织initial cell 原细胞seed deterioration 种子劣变indeterminate 无限性self-perpetuating 自我留存stem cell 干细胞quiescent center 静止中心tunica 原套corpus 原体central zone 中央区peripheral zone 周缘区vegetative meristem 营养分生组织floral meristem 成花分生组织growth periodicity 生长的周期性grand period of growth 生长大周期lagphase 停滞期logarithmic growth phase 指数期linear growth phase 线性期senescence phase 衰减期daily periodicity 昼夜周期性correlation 相关性apical dominance 顶端优势primigenic dominance 原发优势autoinhibition 自动抑制allelopathy 它感作用autotoxicity 自毒photomorphogenesis 光形态建成photoreceptor 光敏受体photosensor 光敏受体phytochrome 光敏色素thermoperiodicity of growth 生长的温周期现象tropic movement 向性运动nastic movement 感性运动phototropism 向光性geotropism 向地性gravitropism 向重力性positive/negative gravitropism 正/负向重力性diagravitropism 横向重力性statolith 平衡石amyloplast 淀粉体chemotropism 向化性hydrotropism 向水性turgor movement 紧张性运动nyctinasty 感夜性thermonasty 感温性seismonasty 感震性action potential 动作电位circadian rhythm 近似昼夜节律ripeness to flower state 花熟状态floral induction 成花诱导floral evocation 成花启动initiation of flower 花的发端floral development 花发育vernalization 春化作用devernalization 去春化作用revernalization 再春化作用vernalin 春化素bolting 抽薹photoperiod 光周期photoperiodism 光周期现象photoperiodic induction 光周期诱导critical dark period 临界暗期critical night 临界夜长florigen 成花素floral determinated state 成花决定态flower bud differentiation 花芽分化hermaphroditic plants 雌雄同株同花植物dioecious plants 雌雄异株植物androecious line 雄性系gynoecious line 雌性系fertility change 育性转化pollenin 花粉素recognition 识别group effect 集体效应parthenocarpy 单性结实phyrin 非丁respiratory climacteric 呼吸跃变dormancy 休眠epistotic dormancy 强迫休眠physiological dormancy 生理休眠after-ripening 后熟stratification 层积处理copigmentation 协同关系作用free radical 生物自由基programmed cell death 程序性细胞死亡apoptosis 细胞凋亡abscission 脱落stress 逆境，胁迫stress physiology 逆境生理strain 胁变escape 避性avoidance 御性tolerance 耐性hydraulic signal 水信号water mass flow 水流hydraustatic pressure 水压chemical signal 化学信号aquaporin 水孔蛋白proteinase inhibitor 蛋白酶抑制剂systemin 系统素electrical signal 电信号osmotin 渗调蛋白chaperone 分子伴侣water stress protein 水分胁迫蛋白heat shock protein 热激蛋白late embryogenesis abundant protein 胚胎发生晚期丰富蛋白lipid transfer protein 类脂转移蛋白kinase-regulated protein 激酶调节蛋白biotic stress 生物逆境pathogenesis-related protein 病程相关蛋白heavy metal binding protein 重金属结合蛋白phytochelatin 植物螯合肽cold-acclimation-induced protein 冷驯化诱导蛋白anaerobic stress protein 厌氧蛋白cross adaptation 交叉适应。