《基因工程》习题及参考答案
一、习题：
1. What are biotechnology and genetic engineering?
2. What is a gene?
3. What are genetically engineered medicines?
4. What do genome research and human genetics deal with?
5. What potentials are held out by genetic diagnosis?
6. What options are given by gene therapy?
7. What is an embryo - and what is a fetus?
8 What is a genetic fingerprint?
9 What does the term "therapeutic cloning" mean?
10 What are stem cells?
11 What is a transgenic organism?
12 What does xenotransplantation mean?
13 How will genetic engineering be used in agriculture?
14 How are genetically modified organisms assessed?
15 What does the German Embryo Protection Act regulate?
16. What is a genome?
17. Is there a risk of bioterrorism?
18. How does genetic engineering affect the environment?
19. Are genetically engineered crops good for farmers?
20.What is the difference between restriction digestion and restriction mapping?
21.Can you combine two different restriction enzymes in the same reaction tubes to
digest the DNA molecules?
22.Why should we need to generate restriction mapping data?
23.How many restriction enzymes available now on the market?
24.Why do you consider mutagenesis in vitro as one of the most critical techniques
for us to understand in genetic engineering class?
25.How do we choose the methods for DNA modification?
26.How do we choose a gene expression system?
27.How can we express eukaryotic gene in E.coli?
28.What should we consider before we start the recombinant protein expression
experiment?
29.What is the advantage of yeast expression system?
30.What is the advantage of insect expression system?
31.Why there are so many different types of vectors available for cloning?
32.What is the difference between cloning vector and expression vector?
33.What is a genetic fingerprint?
34. 基因具体分成多少种类？
35. 什么叫印记基因？
36．什么叫遗传漂变？
37．人类基因组图谱和初步分析结果是在哪一年公布的？
38．人类基因组共有多少基因?
39. 克隆羊成功的技术关键是什么？
40. 有人计划将两个不同物种的动植物体细胞进行融合，然后将融合体的核移植到其中一种生物的未受精卵细胞中，进行体细胞克隆。此计划可行吗？为什么？
41.切口移位标记探针的主要步骤有哪些？
42.用EcoRⅠ和Hind Ⅲ分别切割同一来源的染色体DNA，并进行克隆，在前者的克隆中筛选到A基因，但在后者的克隆中未筛选到A基因，是什么原因？43.在基因工程中，为了在细菌细胞中表达真核细胞的基因产物，为什么通常要用cDNA而不用基因组DNA？为什么要在cDNA前加上细菌的启动子？
44.当两种限制性内切酶的作用条件不同时，若要进行双酶切，应采取什么措施？为什么？
45.有些噬菌体和质粒常常编码一些抗限制性酶的蛋白以中和宿主的限制系统。除此之外噬菌体和质粒还有哪些可能的方式避免宿主的限制系统？
46.为什么大多数内切酶被称为“限制”酶？
47.何谓同裂酶？
48.什么是限制性物理图谱？
49.为什么反转录酶在聚合反应中会出错？
50.RNase A和.RNase H在催化活性和应用上有何不同？
51.切口移位标记DNA前，用DNaseⅠ处理DNA时，应注意什么？
52.DNA连接酶对DNA的复制是很重要的，但RNA的合成一般却不需要连接酶，为什么？
53.RNA聚合酶Ⅲ特异性地转录小分子RNA，但为什么不转录5.8SrRNA?
54.用EcoRⅠ切割外源DNA片段时出现了星号活性，可能是什么原因？
二、参考答案：
1 What are biotechnology and genetic engineering?
Biotechnology is a field of bioscience. In very general terms it can be described as the theory of the use of biological systems in research and applications. Biotechnology applications are a rapidly growing sector of the economy. The number of biotech companies in Germany has increased considerably over the past seven years. Genetic engineering includes all biotechnological processes aimed at effecting specific changes in the genetic material of cells. The principle is, in general, the following: sections of DNA (= deoxyribonucleic acid) from outside sources are introduced into a cell in order to bring about defined changes. What is involved in most cases is the synthesis of molecules, the information for which is contained in the introduced DNA. This process has been used to produce new medicines (see genetically engineered medicines).
2 What is a gene?
A gene is the smallest unit of genetic material. It describes a specific section of DNA which, as a result of its nucleotide (= DNA component) sequence, determines specific functions, characteristics, features, or structures of a cell. The way genes are expressed in each case is dependent on their interaction with other genes and with the environment.
The genome is the entirety of genes contained in an organism. The term is synonymous with genetic material.
3 What are genetically engineered medicines?
In the genetic engineering of medicines genes containing the codes of therapeutically valuable substances are transferred to easily cultivated cells. In most cases bacteria are used, more rarely also yeast cells and the cells of mammals. Medicines produced in this way are called recombinant medicines.
The human insulin introduced (for diabetics) in 1982 was the first recombinant medicine. Since the mid-1990s persons with hemophilia A (the most common form of this blood disease) have been treated to an increasing extent with recombinant factor VIII. Prior to that the only treatment available was factor VIII produced from human plasma.
4What do genome research and human genetics deal with?
Genome research pursues the objective of identifying the structures and functions of the individual genes in the genome. The object of genome research is the study of the genetic material of all organisms, meaning that the genomes of certain plants, animals, and microorganisms are just as much a subject of study as the human genome.
It is an important task of human genome research to identify what genes are involved and in what ways they are involved in causing diseases. This research is expected to bring new approaches in the treatment of cardiovascular diseases, cancer, infectious diseases, and diseases of the central nervous system such as Parkinson's disease, multiple sclerosis, or Alzheimer's disease.
Scientists presented the largely decoded human genome on June 26, 2000. Both scientists and government leaders throughout the world noted this as an epoch-making event and an important milestone in the history of mankind. At the same time this has created new challenges for science, government, and ethics. The field of ethics is called upon to show whether and how this knowledge can be used responsibly in such widely diverse fields as medicine and agriculture.
Human genetics is a medical discipline that deals with human heredity, the causes and diagnosis of hereditary diseases, and the search for possible therapies. Diagnostic procedures today include state-of-the-art genetic diagnoses in addition to traditional analyses of hereditary mechanisms.
5 What potentials are held out by genetic diagnosis?
Modern genetic diagnosis methods make it possible to analyze individual structures of the genome and, in doing so, to identify gene defects associated with hereditary diseases such as cystic fibrosis. This would be beneficial to persons who carry the gene and would run the risk of passing it on to their children. It is conceivable that awareness of an inherited predisposition for a disease that emerges later in life would cause the persons in question to take special precautions such as going in for preventive checkups or pursuing a lifestyle aimed at preventing an outbreak of the disease.
Gene tests can be used to confirm the diagnosis of suspected diseases. More than that, modern methods (such as gene chips) can be used to help a wide range of people, including those who are not sick, to identify genetic characteristics such as predispositions for certain diseases. Here, entirely new ethical and legal questions are arising for society. In the case of predictive gene tests, i.e. tests used to make forecasts as to risks of hereditary illness, it will be a separate task to determine whether these tests will be of value to individuals and to society as a whole.
We need to distinguish between preimplantation genetic diagnosis (PGD), prenata l genetic diagnosis in the context of prenatal care, and postnatal genetic diagnosis. Preimplantation genetic diagnosis (PGD) is a controversial area of genetic diagnosis. After in vitro fertilization PGD can be used to carry out a gene test on an embryo before it is implanted in the mother's uterus. By this means embryos showing a specific genetic defect can be sorted out.
6What options are given by gene therapy?
The objective of gene therapy is to treat or prevent diseases, making use of genetic engineering principles. DNA can, for instance, be introduced into blood or liver cells by means of vectors in order to eliminate genetic defects present there. Or cells that have been genetically modified outside of the body may be implanted in the patient. A further approach is the injection of so-called naked DNA as a kind of vaccine. Naked DNA makes it possible to synthesize proteins against which the vaccinated person builds up an immune reaction as in the case of a conventional vaccination, except that here a much more specifically targeted effect on the immune system is expected. Somatic gene therapy must be distinguished from germ line gene therapy. After germ line gene therapy the effected change in genetic information would be passed on to the offspring of the individual in question. The prerequisite for this would be manipulation of the genetic material contained in an egg cell or a sperm cell or of the genetic material contained in an embryo in the early stages of development. The German Embryo Protection Act imposes a comprehensive ban on carrying out germ line gene therapy in humans.
In the case of somatic gene therapy (= therapy of or involving body cells) only body cells (= somatic cells) are the object of change. Since this type of treatment is directed exclusively at somatic cells, it will not have an effect on the genetic material passed on to the patient's offspring. The predisposition for a given disease will be passed on to the patient's children even if gene therapy is successful. It should be noted that at the present time gene therapy is used mostly to treat malignant cancers, and in Germany exclusively in such cases. The cause of the disease is usually not associated with one single gene but rather with a combination of external factors involving several genes. For part of the diseases treated there is no evidence of a genetic predisposition and in the underlying therapeutic approach the question of a genetic predisposition is without significance.
7 What is an embryo - and what is a fetus?
In human medicine an embryo is usually defined as a fertilized egg cell in the early stages of development up until the point in time when the organs have been fully formed (eighth week of pregnancy).
After that (from the ninth to the thirty-eighth weeks of pregnancy) we speak of a fetus. Sometimes these terms are defined differently from this under the law.
8 What is a genetic fingerprint?
Genetic fingerprinting (also known as DNA fingerprinting) is a procedure provided by molecular biology with which the unique genetic patterns of any individual can be determined. Except in the case of identical twins no two genetic fingerprints are the same.
In paternity suits or in criminal investigations comparisons of DNA fingerprints make it easier or, indeed, may be the only way to identify the guilty party.
9 What does the term "therapeutic cloning" mean?
Cloning is generally understood to be the artificial production of a genetically identical copy of a living organism. The concept refers to complex organisms as well as to individual cells. All the bacteria in a specific colony are genetically identical. In humans this situation is given only in the case of identical twins.
Reproductive cloning is the process of creating genetically identical organisms. For this purpose embryos created artificially, i.e. by means of a nucleus transfer, are implanted in the uterus of the prospective mother where they are to develop into "normal" organisms. The most widely known case of reproductive cloning is Dolly, the sheep that was presented to the public by British scientists in 1997.
The process of cloning consists in removing the (haploid) nucleus from an egg cell and replacing it with the (diploid) nucleus of any given cell of any given origin. This creates the situation in which an embryo and, in the end, a fully developed organism can grow from the egg cell, as in the case of a fertilized egg.
In the case of therapeutic cloning the diploid nucleus comes from the patient in whom destroyed tissue is to be replaced, e.g. following a heart attack or a spinal injury. In
contrast to reproductive cloning, the embryo that has been created artificially by means of a nucleus transfer is not implanted in the uterus of a prospective mother. Instead, in the so-called blastocyst stage of development (about the sixth day) the inner cells are removed from the embryo, destroying it in the process. The stem cells taken from the embryo are used as a raw material for the development of the cell/tissue types needed. The cells or tissues grown from them will not be rejected later when they are transferred to the patient, since they are genetically identical with those already in the patient's body. In contrast to the transplantation of cells and tissues customary today, no medication is needed to suppress defensive reactions of the immune system and the long-term survival of implants becomes feasible. Therapeutic and reproductive cloning in humans is prohibited in Germany under the provisions of the Embryo Protection Act. In the United Kingdom of Great Britain and Northern Ireland therapeutic cloning in humans is now possible under strict regulations, but reproductive cloning continues to be prohibited. It is assumed that many years of research are needed yet before cells or tissues can be transferred successfully in humans on the basis of therapeutic cloning. There has not been enough research carried out thus far on the extent to which the growth of transplanted cells and tissues can be controlled and whether tumors or malignant tissue growth could occur. The cloning of complex organs such as hearts or kidneys is currently beyond reach.
10 What are stem cells?
Stem cells are the predecessors of more differentiated and, as such, more specialized cells. Bonn marrow, for instance, contains stem cells for the various cells that occur in blood such as lymphocytes, granulocytes, or thrombocytes. Stem cells show different degrees of differentiation. They may have the ability to develop into a complete organism, into a variety of different cells, or into specific cells only. These stages are reflections of embryonic development.
In the first days after fertilization embryonic stem cells have what is known as "totipotence", i.e. the ability to develop into a complete organism. In the further
course of development they have varying degrees of "pluripotence", i.e. the ability to develop into specific tissues with specific functions such as liver, kidney, or brain tissue. Small numbers of stem cells are found in these types of tissue in adults or in the blood of the umbilical cords of newborn babies. The latter could possibly serve as an alternative for embryonic stem cells. The extent to which this is the case is currently a subject of research.
Members of the medical community attach considerable hope to stem cell research. They see potential for being able to replace damaged tissues such as myocardial tissue after a heart attack or nerve tissue in the brain in connection with the treatment of Parkinson's disease or in the spinal cord after back injuries. Adult cells cannot be used for this, since they, in contrast to stem cells, are no longer able to integrate themselves into the tissue of an organ and take over the necessary functions.
It is of fundamental importance for implementation in clinical practice to be able to grow unlimited numbers of stem cells at specific levels of differentiation potential and, at the same time, to be able exert precise control over their growth into certain types of cells as needed. Similar to the situation given in connection with therapeutic cloning, questions as to the ability to control tissue growth and potential for the formation of tumors and malignant tissue are still unanswered. There is a need for extensive research here.
11 What is a transgenic organism?
A transgenic organism is an organism, into the genetic material of which exogenous genes have been introduced with the help of genetic engineering methods. This process is also called "genetically engineered change", "gene manipulation", or "germ line modification" in reference to animals. Transgenic organisms pass on the new genetic information to their offspring. Manipulation of human germ lines is prohibited in Germany.
Transgenic animals are being used to a growing extent in medical research, e.g. transgenic mice for research on the Creutzfeldt-Jakob disease (CJD).
Transgenic plants are being developed mostly for use in agriculture. Transgenic microorganisms have been used primarily in the production of medicines and enzymes. Other uses of genetically modified microorganisms include food processing and the production of biodegradable plastics. In some cases transgenic animals, plants, and microorganisms have been patented.
12 What does xenotransplantation mean?
Xenotransplantation means the transplantation of animal organs, tissues, or cells into humans as well as exposure to animal organs or cells through the bloodstream. Although primates were originally considered to be suitable donors for xenotransplantation, pigs are now considered to be a better choice for a number of reasons.
The transplantation of entire organs from pigs is prevented by uncontrollable hyperacute reactions of the human immune system. The blood vessels of the transplanted organ clog up within a few minutes and the organ dies. An attempt is being made to create donor animals genetically engineered to reduce or entirely eliminate the rejection of transplants by the immune system. At present it is not yet possible to say whether xenotransplantation will become a clinical reality.
There are fundamental reservations with regard to xenotransplantation. They derive from the fear that new types of infectious pathogens could develop from animal cells or organs. Infections of this kind might not be limited to the individuals concerned and spread throughout the population. The probable need to suppress immune reactions to the transplant that would be stronger than in the case of an allotransplantation (i.e. human to human) would tend to favor the occurrence of infections.
13How will genetic engineering be used in agriculture?
When genetically modified organisms are developed for use in agriculture we speak of "green biotech". Their use usually means a large-scale presence in the environment and needs to be approached carefully for this reason. Consumer acceptance of green
biotech has been low thus far and the use of genetic engineering in animal husbandry raises ethical questions.
Genetic engineering is being used in plant breeding to create disease and pest-resistant plants and, in this way, reduce the use of pesticides. There is a need, in particular, to monitor the long-term effects of genetically altered organisms on the environment, since this cannot be fully determined in advance on the basis of experiments subjected to limitations with regard to time and geography. There is also a need for long-term monitoring after admission of genetically modified organisms to the market. It is only in this way that it will be possible to identify possible undesirable long-term effects of their use on a large scale and to obtain reliable data for evaluation.
Objectives being pursued with the help of genetic engineering in the field of agriculture:
Studies are being carried out on the use of genetic engineering
* to produce pest and disease resistance, i.e. fungus resistance, bacteria resistance, virus resistance and insect resistance in plants
* for biological pest control making use of genetically engineered viruses
* to produce herbicide resistance in plants (worldwide the most common genetic change being made in crop plants)
* to enhance the quality of plant products, e.g. improve the shelf life, storage life, digestibility, nutritional value, and taste of foods
* to produce feeds
* to produce regenerative raw materials. Genetic engineering has the potential to broaden the range of regenerative resources produced for the chemicals, cosmetics, and pharmaceuticals industries, to broaden the range of raw materials available for energy consumption, or to make applications of this kind possible, and, as such, to supplement the petroleum resource base.
* to produce developments in the breeding and raising of livestock aimed at reducing costs by means of higher fattening performance, lower material consumption, improved profitability, the development of new products, or the development of disease resistance.
A process of rethinking the foundations and conditions of food production has begun, triggered by the BSE crisis. The German government takes the view that it is only through a systematic, consumer-oriented reorientation of agricultural policy that it will be possible to restore the confidence of the public in food production.
The objective pursued in connection with the reorientation of agricultural policy will be to give a convincing response to the increased requirements of society with regard to health and environmental protection with a consumer-oriented agricultural policy and, in doing so, to include the conscionable potentials of green biotech.
14 How are genetically modified organisms assessed?
Approval regulations and assessment standards exist for genetically modified organisms under the Genetic Engineering Act. Under the provisions of the Genetic Engineering Act characteristics of genetically modified organisms must be studied and their interactions with the environment must be predicted and evaluated before they can be intentionally released into the environment. On this basis it is decided whether approval can be given for test releases or for their marketing (commercial trade). When genetically modified organisms are handled in experimental facilities (e.g. labs, fermenters) for research or production purposes, they, along with the research work or production processes being carried out, are assessed with regard to their safety, and the experimental facility in question is subject to licensing approval. The EU member countries and the European Parliament adopted a fundamental amendment to Council Directive 90/220/EEC on the deliberate release into the environment of genetically modified organisms. The new directive entered into force at the end of February 2001. Under the new directive it will be possible to combine the approval of genetically modified organisms for market consumption with binding regulations on monitoring the GMO in question. In addition, general monitoring of genetically modified organisms will be introduced. Both the monitoring of specific GMOs released into the environment as well as general monitoring are aimed at determining and assessing environmental changes and possible effects on human health. In particular, knowledge gaps on environmental effects are to be closed.
15 What does the German Embryo Protection Act regulate?
The Embryo Protection Act entered into force on January 1, 1991.
It contains the following elements enforceable under criminal law:
* a ban on the illegal application of procedures used in reproductive medicine,
* the protection of human embryos against illegal use,
* a ban on artificial fertilization of egg cells with the sperm of persons who are dead, * a ban on gender selection,
* protection against arbitrary fertilization and transfer of embryos,
* a ban on gene transfers in germ cells,
* a ban on cloning, and
* a ban on the creation of chimeras and hybrids.
Cartagena Protocol on Biosafety
The Second Conference of the Parties to the Convention on Biological Diversity held in Jakarta from 6 to 17 November 1995 adopted a decision to formulate a supplementary protocol to the convention which has come to be known as the Cartagena Protocol on Biosafety. After long and difficult negotiations this legally binding protocol was adopted in Montreal in January 2000 and since then has been signed by about 80 countries. The protocol contains provisions on the safe handling and transfer of living modified organisms or LMOs (synonymous with genetically modified organisms or GMOs). Particularly important in this context are the provisions on transboundary movement of LMOs. Exports for experimental release and transfer of LMOs are to take place only if and when the receiving state has been informed in advance and in detail of the risks involved, in particular of risks for biological diversity, and has given its approval. There is also a provision requiring that biotech accidents be reported if transboundary effects are to be reckoned with.
In the framework of the Convention on Biological Diversity agreements were concluded on the handling of biotechnology and the distribution of its benefits. On the basis of these agreements a Clearing House Mechanism (CHM) was established which serves as a liaison office for information on biological diversity. It also collects
information on the biological safety regulations that exist in the countries who are parties to the Convention on Biological Diversity, detailed reports and articles on biosafety-related questions and issues, as well as the latest news items and scheduled meetings.
16. What is a genome?
The standard definition of a genome is that it is the whole collection of the genes of an organism. However, a genome is much more. It is not only the set of all genes present in the nucleus of the cells of the organism, but also the way these genes are organised next to each other. If you consider a recipe for your favourite dish, you can easily understand that having only the list of ingredients would not be enough to allow you to prepare the dish! A genome is the whole text of the DNA present in every cell of a living organism. It therefore comprises regions corresponding to CDSs, as well as all the regions in between ("intergenic regions").
The genome organisation is so important that in fact one can often tell something about the order of the genes in the genome, genes with related functions being often in some sort of proximity.
17. Is there a risk of bioterrorism?
This text was written at the creation of the Centre, well before the terrorists attacks using anthrax, demonstrating, alas, that our world is not as good as it should be.... Unfortunately, human beings are often enflamed by wrong or even plainly destructive ideas. This gives rise to aggressive behaviour. Biological warfare existed for a long time. The Spanish invadors of America used it, and, more recently, there have been quite a few attempts in China by Japanese militaries. One cannot, therefore, dispell the idea as far-fetched. However, it must be understood that biological warfare, in contrast to all other means used in wars, is difficult, if not impossible, to control once it has been launched. In particular, agents such as Bacillus anthracis, which have been used for that purpose, remain in the environment for decades or even centuries. A test bomb, exploded by the British in Gruinard Island, and full of B. anthracis spores,
resulted in contamination of the island for 50 years, and one had to scrape the surface of its soil and spread strong bactericides to get rid of most of the spores! This does not mean of course that this type of warfare will not be used, but it is clearly a very mad way to proceed. Please note here the symptoms of anthrax. The Institut Pasteur in Paris provides a large sum of information on the topic (in French).
Can we trust the sanity of human beings? To counteract this possibility the best is certainly to control as much as possible information on the matter, and refrain to spread it. Clearly, freedom does not mean freedom to kill. Misinformation is another possibility, but it is difficult to control.
18. How does genetic engineering affect the environment?
Unlike previous pollution genetically engineered crops are living organisms, they can reproduce and spread. This poses an ongoing threat to the environment. If something goes wrong this GE pollution cannot simply be recalled or cleaned up.
Ecologists believe that the release of these unpredictable organisms could have far reaching consequences in much the same way that non-native species such as the cane toad have become pests. Such effects may only emerge after a few generations. Persistent GE crops may cross with related weeds to become superweeds? Particularly vigorous weeds that out-compete other plants and destroy natural plant ecosystems. GE crops produce seed and pollen which can contaminate other crops and also soil. Soil is extremely complex and there is already some evidence that GE crops have affected the fertility of soil.
One type of GE crops designed to produce their own insecticide have been shown to be harmful to the larvae of butterflies, to ladybirds and other important insects. Beekeepers are concerned that such crops may also be harmful to bees who ingest pollen. The real impact of GE crops on the many types of insect and wildlife found in the environment has not yet been checked.
So far most of the GE crops that are grown are designed to be used with powerful weedkillers. When the Roundup Ready canola? It is sprayed with roundup weedkiller
the plant lives but all other plants die, taking away shelter and food for many insects and birds depending on these wild plants. This too threatens natural systems.
19 Are genetically engineered crops good for farmers?
In the USA and Canada, where GE crops have now been grown for 5 years, a coalition of 33 farm and agriculture groups recently issued a warning that genetic engineering in agriculture has significantly increased the economic uncertainty of family farmers throughout the U.S. and the world. ?
The agrochemical companies that produce GE seeds require farmers to sign legal agreements specifying how to farm and promising not to save seed. They also expect farmers to pay royalties. Companies such as Monsanto then aggressively sue farmers who they believe are using their seeds without signing such agreements. Unfortunately due to contamination many farmers are finding they have GE crops on their land whether they asked for it or not. In Canada, Monsanto sued a canola grower called Percy Schmeiser because GE canola was growing on his land as a result of contamination. Even though Schmeiser did not want the contamination Monsanto argued successfully that he owed them money anyway.
Conventional growers are discovering that GE crops from neighbouring fields have become weeds that cannot be sprayed off with herbicides because they have inbuilt resistance. The Royal Society of Canada has warned that most of the country's prarie land is now contaminated with herbicide-resistant canola weeds, the removal of which raises farm costs.
At the end of harvest US and Canadian farmers are discovering a further problem of GE crops that export markets. Following the introduction of GE canola in Canada, sales to Europe dried up. The same is true of US corn which is no longer sold to Europe and has lost important markets in Asia.
20.What is the difference between restriction digestion and restriction mapping? Restriction digestion is meant to the biochemical reaction using restriction enzymes to cut DNA into smaller pieces before electrophoresis; and restriction mapping is a
technique used to determine the location of restriction sites in a DNA molecule.
21.Can you combine two different restriction enzymes in the same reaction tubes
to digest the DNA molecules?
Y es, you can.
But you need to be very careful to select the combination of different enzymes. The critical point of consideration is the reaction buffer. The ion requirement for individual enzyme is different from each other, and sometime you have to cut the DNA molecule sequentially, which means you have to add an extra purification step between two digestions.
22.Why should we need to generate restriction mapping data?
Restriction mapping data is essential for cloning and DNA sequencing. Y ou have to choose specific restriction site for basic cloning before you can start for DNA sequencing or other DNA manipulation.
23.How many restriction enzymes available now on the market?
Now there are hundreds restriction enzymes available on the market. The longer the DNA sequences of the restriction enzymes can recognize, the better for DNA cloning, especially the 8-base recognition enzymes.
24.Why do you consider mutagenesis in vitro as one of the most critical
techniques for us to understand in genetic engineering class?
The technology of mutagenesis in vitro serves the molecular biologists as a tool to manipulate DNA, explore, modify and enhance the function of proteins by base pair change. Basically most of commercialized protein products are modified one, even including pharmaceutical drugs.
25.How do we choose the methods for DNA modification?
I think you should make your decision based on your experimental plan, namely what do you want to modify the target gene you are working on.
26.How do we choose a gene expression system?
1)choose the gene expression system by your experimental plan;
2)choose the gene expression system by the nature of the target gene;
3)choose the gene expression system by the convenience in the lab.
27.How can we express eukaryotic gene in E.coli?
There six steps involved:
First, isolate the gene you are interest; 2nd, you need to clone it into proper expression vector; 3rd, transform the recombinant molecule into host cells which matches the vector you used in DNA cloning; 4th, pick up the desired colonies by selection; 5th, grow cells through fermentation;
And the last you should isolate and purify the protein product.
28.What should we consider before we start the recombinant protein expression
experiment?
1). Prokaryotic and eukaryotic promoters and translation signals are
different...they are not exchangeable;
2). Processing also presents a problem for bacterial expression of human mRNAs;
3). Post-translational modifications can be important for protein function
29.What is the advantage of yeast expression system?
1)Easier and less expensive than higher eukaryotic cells
2)Rapid growth on inexpensive media
3)Ideal for large-scale production of heterologous proteins
4)Often comfortable with genetic manipulation
5)Exhibit near-native conformation, PTMs, processing
6)Well-defined secretory pathways for extracellular export of the recombinant
gene product
7)Usually safe to use
30.What is the advantage of insect expression system?
1)Produce proteins that has PTMs similar to mammalian systems
2)Often properly folded and functional
3)Ideal for producing moderate to high levels of eukaryotic proteins for
structure-function assays
31.Why there are so many different types of vectors available for cloning? Genetic engineering develops very fast, demands drives vectors’ evolving.
For different purposes, we need different types of vectors. Basically we can divide vectors into two categories: cloning vectors and expression vectors. But expression vectors now can be used for cloning too. The latest development of vector is the invention of BAC (bacterial artificial chromosome) and Y AC (yeast artificial chromosome). They are very helpful for speed up of human genome sequencing project.
32.What is the difference between cloning vector and expression vector?
Expression vector needs all the necessary components which cloning vectors needed for replication in a bacterial culture.
Apart from that, expression vector also needs: 1). A specific promoter to drive expression of the gene; 2). ribosome binding site; 3). Termination signals for transcription and translation; 4). Selection marker for host cells of expression.
33.What is a genetic fingerprint?
Genetic fingerprinting (also known as DNA fingerprinting) is a procedure provided by molecular biology with which the unique genetic patterns of any individual can be determined. Except in the case of identical twins no two genetic fingerprints are the same.
In paternity suits or in criminal investigations comparisons of DNA fingerprints make it easier or, indeed, may be the only way to identify the guilty party.
34. 基因具体分成多少种类？
答：分子遗传学证明基因是有各种不同的功能结构的。由于结构和功能的不同，现代生命科学将基因分为很多种：
（1）结构基因，即对蛋白质的氨基酸组成和顺序起决定作用的基因，它携带遗传信息通过转录、翻译等过程得以表达。
（2）调控基因，即可以控制结构基因表达的基因，常位于结构基因的邻近位置，通过活性状态的改变来影响结构基因的活性。
（3）跳跃基因，即可以在DNA上跳动，甚至在不同的染色体上移动的基因。（4）断裂基因，即在基因结构中插入与氨基酸编码无关的DNA间隔区从而使一个基因被分隔成不连续的若干区段的基因，多存在于真核生物中。
（5）重叠基因，即多个基因共用碱基对的基因，在一些病毒和噬菌体的DNA上发现有这种现象。重叠方式有完全重合叠，也有部分重叠。
（6）重复基因，即在基因组中有多个拷贝的基因。在真核生物基因组中发现这种现象，真核生物中的重复基因可以达到30%。重复基因主要是为了满足生物体快速发育的需要。
（7）假基因，即与正常功能基因顺序基本相同却不具有控制蛋白质合成的功能的基因，在真核生物中是很普遍存在，它形成的主要原因是小的碱基对缺失或插入以致不能正常编码。
35. 什么叫印记基因？
答：来自于不同亲本的等位基因表达不同的现象。
36．什么叫遗传漂变？
答：由于样本的机误导致群体基因频率的随机改变。
37．人类基因组图谱和初步分析结果是在哪一年公布的？
答：六国于2001年公布了人类基因组图谱和初步分析结果。
38．人类基因组共有多少基因?
答：人类基因组含有3万多个基因。
基因工程试题 一、名词解释(4 ‘*10) 1.基因工程技术：按照人们的愿望，进行严密的设计，通过体外DNA 重组和转移等技术，有目的地改造生物种性，使现有物种在较短的时间内趋于完善，创造出新的生物类型。 2.感受态细胞：受体细胞经一些特殊方法(如CaC12、RbCl (KC1) 等化学试剂)处理后，细胞膜的通透性发生了暂时性改变，成为能允许外源DNA 分子进入的细胞状态。 3.cDNA文库：从组织细胞中分离得到纯化的mRNA,然后以mRNA为模板，利用逆转录酶合成其互补DNA,再复制成双链cDNA片段，与适当载体连接后导入受体菌内，扩增，构建cDNA文库。 4.鸟枪法：指将某种生物体的全基因组或单一染色体切成大小适宜的 DNA 片段，分别连接到载体DNA上，转化受体细胞，形成一套重组兑隆，从中筛选出含有目的基因的期望重组子。 5.SD序列(Shine-Dalgarno):位于翻译起始密码了•上游的6-8个核苷酸序列(5’ UAAGGAGG 3’)，它通过识别大肠杆菌核糖体小亚基中的16S rRNA 3’端区域3’ AUUCCUCC 5’并与之专一性结合，将mRNA定位于核糖体上，从而启动翻译。 6.包涵体：胞内高效表达时，工程菌因大景合成异源蛋白质所形成的水不溶性积聚物。
7.复合转座子：是由两个重复序列夹着一个或多个结构基因如某些抗药性基因和其它基因组成。存在于R因子及其它质粒中。复合转座子两端的组件由IS和类IS组成。 8.菌落原位杂交：是将菌落或噬菌斑转到固相膜上，原位裂解细胞后使核酸固定在膜上，然后与探针杂交。用标记的核酸探针，经放射自显影或非放射检测体系，在组织细胞间期染色体上对核酸进行定位和相对定量研究的一种手段。 9.RACE：是一种通过PCR进行cDNA末端快速克隆的技术，是以mRNA 为模板反转录成cDNA第一链后用PCR技术扩增出某个特异位点到3, 或5，端之间未知序列的方法。 10.连续式发酵：即在连续发酵反应器中，细胞的总数和培养液总体积同时维持恒定，前提是由培养液流出所造成的细胞损失正好为细菌分裂所产生的新细胞弥补。 二、简答题(6’ *5) 1、一个理想克隆载体应具备的条件有哪些？ 答：⑴分子较小，可携带比较大的DNA片段。 (2)能独立于染色体而进行自主复制并且是高效的复制。 (3)要有尽可能多种限制酶的切割位点，但每一种限制酶又要最少的切割位点(多克隆位点multiple cloning sites, MCS)。
第二章 1. 名词解释：核酸内切酶、核酸内切限制酶、同裂酶、同尾酶、核酸外切酶、末端脱氧核苷酸转移酶 答： 核酸内切酶：是一类从多核苷酸链的内部催化磷酸二酯键断裂的酶。 核酸内切限制酶：是一类能够识别双链DNA分子中的某种特定核苷酸序列（4—8bp），并由此处切割DNA双链的核酸内切酶。 同裂酶：识别位点的序列相同的限制性内切酶。 同尾酶：识别的序列不同，但能切出相同的粘性末端。 核酸外切酶：是一类从多核苷酸链的一头开始催化降解核苷酸的酶。 末端脱氧核苷酸转移酶：可以不需要模板，在单链DNA或突出的双链DNA 3’-OH端随机 添加dNTPs的酶 2. 限制性内切核酸酶的命名原则是什么？ 答：限制性内切核酸酶按属名和种名相结合的原则命名的，即：属名+种名+株名+序号； 首字母：取属名的第一个字母，且斜体大写； 第二字母：取种名的第一个字母，斜体小写； 第三字母：(1)取种名的第二个字母，斜体小写； (2)若种名有词头，且已命名过限制酶，则取词头后的第一字母代替。 第四字母：若有株名，株名则作为第四字母，是否大小写，根据原来的情况而定，但用正体。 顺序号：若在同一菌株中分离了几种限制酶，则按先后顺序冠以I、Ⅱ、Ⅲ、…等，用正体。 3．部分酶切可采取的措施有哪些？ 答：1）缩短保温时间 2）降低反应温度 3）减少酶的用量 4. 在序列5'-CGAACATATGGAGT-3'中含有一个6bp 的Ⅱ类限制性内切核酸酶的识别序列，该位点的序列可能是什么? 答：回文序列是：5'-CATA TG-3， 5．什么是限制性内切核酸酶的星号活性? 受哪些因素影响? 答：Ⅱ类限制酶虽然识别和切割的序列都具有特异性，但是这种特异性受特定条件的限制，即在一定环境条件下表现出来的特异性。条件的改变，限制酶的特异性就会松 动，识别的序列和切割都有一些改变，改变后的活性通常称第二活性，而将这种因 条件的改变会出现第二活性的酶的右上角加一个星号表示，因此第二活性又称为星 活性。 概括起来，诱发星活性的因素有如下几种：(1)高甘油含量(>5％, v／v)；(2)限制性 内切核酸酶用量过高(>100U／ugDNA)；(3)低离子强度(<25 mmol／L)；(4)高pH(8．0 以上)；(5)含有有机溶剂，如DMSO，乙醇等；(6)有非Mg2+的二价阳离子存在(如 Mn2+，Cu2+，C02+，Zn2+等)。 第三章 1．如何将野生型的λ噬菌体改造成为一个理想的载体? 答：①删除λ噬菌体的非必需区，留出插入空间；并在余下的非必须区内制造限制酶切点 ②引进某些突变表型，作为选择标记 ③突变某些基因，使它成为安全载体 ④删除λDNA必须区段上常用的限制酶切点
第二章习题 一、单选题 1.在基因操作中所用的限制性核酸内切酶是指（ B ） A．I类限制酶 B. II类限制酶 C. III类限制酶 D.核酸内切酶 E. RNAase 2.下列关于同裂酶的叙述错误的是( B ) A. 是从不同菌种分离到的不同的酶,也称异源同工酶。 B. 它们的识别序列完全相同。 C. 它们的切割方式可以相同，也可以不同。 D. 有些同裂酶识别的完整序列不完全一样，但切割位点间的序列一样。 E. 两种同裂酶的切割产物连接后，可能会丢失这两个同裂酶的识别位点。 3. 多数限制酶消化DNA的最佳温度是（ A ） A. 37℃ B.30℃ C.25℃ D.16℃ E.33℃ 4. 下列关于限制酶的叙述错误的是( B ) A. I类限制酶反应需要 Mg2+、ATP和S-腺苷蛋氨酸。 B. II类限制酶反应需要Mg2+、ATP。 C. III类限制酶反应需要Mg2+、ATP，S-腺苷蛋氨酸能促进反应，但不是绝对需要。 D. I、III类限制酶对DNA有切割和甲基化活性，II类限制酶对DNA只有切割活性而无甲基化活性。 E. II类限制酶要求严格的识别序列和切割点，具有高度精确性。 5. 如果一个限制酶识别长度为6bp ,则其在DNA上识别6bp的切割概率为( D ) A. 1/44 B. 1/66 C. 1/64 D.1/46 E. 1/106 6. 多数II类限制酶反应最适PH是 ( C ) A. PH:2-4 B. PH:4-6 C. PH:6-8 D. PH:8-10 E. PH:4-10 7. 下列关于限制酶反应的说法错误的是 ( D ) A. 限制酶识别序列内或其邻近的胞嘧啶、腺嘌呤或尿嘧啶被甲基化后，可能会阻碍限制酶的酶解活性。 B. 许多限制酶对线性DNA和超螺旋DNA底物的切割活性是有明显差异的。 C. 有些限制酶对同一DNA底物上不同酶切位点的切割速率会有差异。 D. 限制酶反应缓冲系统一般不用磷酸缓冲液，是由于磷酸根会抑制限制酶反应。 E. BSA对许多限制酶的切割活性都有促进作用，所以酶切反应中常加入一定量的BSA。 8. II类限制酶反应中必须的阳离子是（ C ）
作业一： 一、名词解释： 1、基因：是遗传的物质基础，是DNA（脱氧核糖核酸）分子上具有遗传信息的特定核苷酸 序列的总称，是具有遗传效应的DNA分子片段。 2、基因组该指单倍体细胞中包括编码序列和非编码序列在内的全部DNA分子 3、操纵子:原核生物的几个功能相关的结构基因往往排列在一起，转录生成一个mRNA，然 后分别翻译成几种不同的蛋白质。这些蛋白可能是催化某一代谢过程的酶，或共同完成某种功能。这些结构基因与其上游的启动子，操纵基因共同构成转录单位，称操纵子。 4、启动子:是RNA聚合酶结合位点周围的一组转录控制组件，包括至少一个转录起始点。在 真核基因中增强子和启动子常交错覆盖或连续。有时，将结构密切联系而无法区分的启动子、增强子样结构统称启动子。 5、增强子:是一种能够提高转录效率的顺式调控元件，最早是在SV40病毒中发现的长约20 0bp的一段DNA，可使旁侧的基因转录提高100倍，其后在多种真核生物，甚至在原核生物中都发现了增强子。增强子通常占100～200bp长度，也和启动子一样由若干组件构成，基本核心组件常为8～12bp，可以单拷贝或多拷贝串连形式存在。 6、基因表达：是指细胞在生命过程中，把储存在DNA顺序中遗传信息经过转录和翻译，转 变成具有生物活性的蛋白质分子。 二、简答题 1、说明限制性内切核酸酶的命名原则要点。 答：限制性内切核酸酶采用三字母的命名原则,即属名+种名+株名的各一个首字母,再加上序号. 基本原则: 3-4个字母组成,方式是:属名+种名+株名+序号; 首字母: 取属名的第一个字母,且斜体大写;第二字母: 取种名的第一个字母,斜体小写;第三字母: (1)取种名的第二个字母,斜体小写;(2)若种名有词头,且已命名过限制酶,则取词头后的第一字母代替.第四字母: 若有株名,株名则作为第四字母,是否大小写,根据原来的情况而定,但用正体. 顺序号: 若在同一菌株中分离了几种限制酶,则按先后顺序冠以I,Ⅱ,Ⅲ,…等,用正体. 2、什么是限制性内切核酸酶的星号活性？受哪些因素影向？ 答：Ⅱ类限制酶虽然识别和切割的序列都具有特异性，但是这种特异性受特定条件的限制，即在一定环境条件下表现出来的特异性。条件的改变，限制酶的特异性就会松动，识别的序列和切割都有一些改变，改变后的活性通常称第二活性，而将这种因条件的改变会出现第二
【基因工程复习题及参考答案】 绪论、基因工程基础 一、填空题 1. 基因工程是20 世纪70 年代发展起来的遗传学的一个分支学科。基因工程技术的诞生使人们从简 单地利用现存的生物资源进行诸如发酵、酿酒、制醋和酱油等传统的生物技术时代，走向按人们 的需要定向地改造和创造具有新的遗传性品种的时代。 2．Cohen 等在1973 年构建了第一个有功能的重组DNA 分子。 3．基因工程的两个基本特点是：（1）分子水平上的操作，（2）细胞水平上的表达。4．基因克隆中三个基本要点是：克隆基因的类型、受体的选择和载体的选择。 5．克隆基因的主要目的有四个：①扩增DNA ；②获得基因产物；③研究基因的表达调控； ④改造生物的遗传特性。 6．基因工程的基本程序包括DNA 制备、体外DNA 重组、遗传转化、转化细胞的筛选等方面。 7．基因工程又称重组DNA 技术/基因操作技术/基因克隆/分子克隆/遗传修饰/新遗传学。8．克隆的进行依赖于各种参与DNA 新陈代谢的酶的使用，它们主要是从原核细胞中分离的。限制 性内切核酸酶能够将DNA 切割成特定大小的片段。各种各样的DNA 聚合酶也是克隆所必需的， 包括将RNA 转录出单链DNA 的反转录酶。DNA 连接酶用于连接限制性片段。克隆也需要来 源于微生物的DNA 序列，包括用来运载被被克隆DNA 的克隆载体。质粒载体来源于天然细菌 质粒，通常携带有抗生素抗性基因。其他载体来源于噬菌体，如λ噬菌体，它们经过修饰后可以 运输外源DNA。要想克隆一个感兴趣的真核生物的基因就必须先制作一个能与该基因杂交的探 针。人们已经发展了很多有效而精细的技术来分析克隆的DNA。例如，极微量的DNA 片段可以 通过PCR 得到扩增。在感兴趣基因的启动子上连接，如CAT 或LacZ 之类的报告基因 能够定 量检测基因的活性。 二、选项题（单选或多选） 1．因研究重组DNA 技术而获得诺贝尔奖的科学家是（C ）。 A. Kornberg A B. Gilbert W C. Berg P D. McClintock 2．第一个用于构建重组体的限制性内切核酸酶是（A ）。 A. Eco R I B. Eco B C. Eco C D. Eco R II 3. 靶（目的）基因通常是指（A ）。
答案：基因操作。将在细胞外产生的核酸（物质）分子插入到病毒，质粒或其它载体系统中，再整合到特定的宿主中，从而形成一种新的可连续繁殖的有机体。名词解释问题： Interrupted genes 参考答案：间断基因。序列中间插入有与氨基酸编码无关的 DNA 间隔区，使一个基因分隔成不连续的若干区段。我们称这种编码序列不连续的基因为间断基因。名词解释问题： Promotor 参考答案：启动子。 DNA 分子可以与 RNA 聚合酶特异结合的部位，也就是使转录开始的部位。 名词解释问题： Subcloning 参考答案：亚克隆。当初始克隆中的外源 DNA 片段较长，含有许多目的基因以外的 DNA 片段时，在诸如表达、序列分析和突变等操作中不便进行，将目的基因所对应的一小段 DNA 找出来，这个过程叫“亚克隆”。 填空题问题：基因操作（Gene manipulation）的核心部分是基因克隆（gene cloning）， gene cloning 的基本要点有（），（）和（）。 参考答案：克隆基因的类型；受体的选择；载体的选择 填空题问题：（）是遗传物质的基本单位,也是作为遗传物质的核酸分子上的一段片段。它可以是连续的，也可以是（）；可以是（），也可以是RNA；可以存在于染色体上，也可以（） 参考答案：基因；连续；DNA；存在于染色体之外（如质粒，噬菌体等） 填空题问题：基因操作并不是一个法律概念，除了包括基因克隆外，还包括基因的（）、（）、（）和（）等与基因研究相关的内容。 参考答案：表达；调控；检测；改造 填空题问题：启动子（Promotor）是指（）。通常一个 Gene 是否表达，（）是关键的一步，是起决定作用的。在转录过程中，Promoter还是（）的结合位点。参考答案：基因转录过程中控制起始的部位；转录起始；RNA 聚合酶 填空题问题：原核生物的 RNA polymerase 主要由两部分组成：（）和（），其中σ-因子并不参与 RNA 的合成，它的作用主要是（）。 参考答案：核心酶；σ因子；识别要转录的起始位点 填空题问题：从（）到（）的这一段距离称为一个转录单位，或者一个转录产物，其中可包括一个或者多个 Gene 。 参考答案：启动区；终止区 填空题问题：转录终止子主要有两种，一种是（），另一种是（）。 参考答案：依赖ρ因子；不依赖ρ因子 填空题问题： Gene 的书写方式，通常是写出的 DNA 序列总是与（）相同的那条链，方向是从（）到（）。对于碱基的位置，一般自转录起始点向两个方向编号，其中转录起始点定为（），（）定为 -1 。 参考答案： mRNA；5'；3'；+1；在起始点上游第一个碱基 填空题问题：重叠基因（Overlapping gene）是指（），间隔基因（Interrupted gene）是指（） 参考答案：一个基因的序列中，单个 DNA 序列可编码一个以上的蛋白质；在编码序列中间插入的一段无编码作用的碱基序列 简答题问题：简述基因克隆的两个基本特征？ 参考答案：基因克隆的两个基本特征是一是强调外源核酸分子（一般情况下都是 DNA）在不同宿主中的繁殖，打破自然种的界限将来自于不相关物种的基因放入一个宿主中是基因操作的一个重要特征，基因操作的另一个重要特征是繁殖。 简答题问题：谈谈你对 gene 的认识，并简要说说 gene 概念的发展情况？ 参考答案：能够表达和产生基因产物（Proteinor RNA）的 DNA 序列（可自由发挥，发展情况请查找相关资料）基因概念的发展：基因作为遗传学中的专用术语，其概念每发展一步都意味着遗传学乃至整个生物学的一次革命和突破，其内容的每次丰富和充实都凝聚着生物学家们的滴滴心血。“基因”概念的提出：遗传学的奠基人孟德尔在 1866 年发表的论文《植物杂交试验》指出生物每一个性状都是通过遗传因子来传递的，遗传因子是一些独立的遗传单位。这样把可观察的遗传性状和控制它的内在的遗传因子区分开来了，遗传因子作为基因的雏形名词诞生了。 1909 年丹麦遗传学家约翰逊在《精密遗传学原理》一书中提出“基因”概念，以此来替代孟德尔假定的“遗传因子”。从此，能够表达和产生基因产物（protein or RNA）的 DNA 序列（可自由发挥）“基因”一词一直伴随着遗传学发展至今。基因结构和功能的探索：摩尔根和他的学生们利用果蝇作了大量的潜心研究， 1926 年他的巨著《基因论》出版，从而建立了著名的基因学说，首次完成了当时最新的基因概念的描述，即基因以直线形式排列，它决定着一个特定的性状，而且能发生突变并随着染色体同源节段的互换而交换，它不仅是决定性状的功能单位，而且是一个突变单位和交换单位。 1941 年比德尔和塔特姆提出一个基因一个酶说； 1949 年鲍林与合作者在研究镰刀型细胞贫血症时推论基因决定着多肽链的氨基酸顺序； 1944 年艾弗里、麦卡蒂首次用实验明确证实： DNA 是遗传信息的载体； 1952 年赫尔希和蔡斯进一步证明遗传物质是 DNA 而不是蛋白质； 1953 年美国分子生物学家沃森和英国分子生物学家克里克通力协作，根据 X 射线衍射分析，提出了著名的 DNA 双螺旋结构模型，进一步说明基因成分就是 DNA ，它控制着蛋白质的合成； 1957 年法国遗传学家本滋尔以 T4 噬菌体作为研究材料分析了基因内部的精细结构，提出了顺反子学说。这个学说打破了过去关于基因是突变、重组、决定遗传性状的“三位一体”概念及基因是最小的不可分割的遗传单位的观点，从而认为基因为 DNA 分子上一段核苷酸序列，负责着遗传信息的传递，一个基因内部仍可划分为若干个起作用的小单位，即可区分成顺反子、突变子和重组子。一个作用子通常决定一种多肽链合成，一个基
高中生物选修三“基因工程”综合练习 (20题含答案) 请回答下列有关克隆技术和基因编辑的问题： 1）克隆技术是指通过人工手段复制出与原体完全一样的 生物体。目前，克隆技术主要分为三种：体细胞核移植克隆、胚胎分裂克隆和基因克隆。其中，最常见的克隆方法是。 2）基因编辑技术是指通过改变生物体的基因序列来实现 对其性状的调控。CRISPR/Cas9系统是目前最常用的基因编辑技术之一，其中CRISPR指的是，Cas9则是。 3）基因编辑技术的应用非常广泛，其中包括疾病基因治疗、农业生产、生物安全等领域。以疾病基因治疗为例，基因编辑技术可以通过修复或替换患者体内的有缺陷基因，从而达到治疗疾病的目的。目前，基因编辑技术已经被用于治疗包括血友病在内的多种疾病。 答案：（1）体细胞核移植克隆（1分）（2）“类细菌重 复间隔簇”、“CRISPR相关蛋白9”（1分）（3）血友病（1分）逆转录病毒载体是目前应用较多的动物基因工程载体之一。它可以将外源基因插入到受体细胞染色体中，使外源基因随染色体DNA一起复制和表达。研究发现逆转录病毒基因组
（RNA）的核心部分包括三个基因：gag基因（编码病毒的核 心蛋白）、pol基因（编码逆转录酶）、env基因（编码病毒 的表面糖蛋白）。位于这些基因的两端有LTR序列（含有启 动子、调节基因等），控制着逆转录基因组核心基因的表达及转移。下图展示了构建逆转录病毒载体及用于培育转基因小鼠的过程。 1）逆转录病毒的遗传信息储存在RNA中，其中包含四 种核苷酸排列顺序中的四种脱氧核苷酸。 2）过程②中，外源基因一定要插入到病毒DNA的LTR 序列中的启动子后，其主要目的是控制外源基因的表达。 3）过程③中，导入病毒蛋白编码基因（gag，pol和env）的目的是合成组成病毒的蛋白质。 4）过程④中，逆转录病毒载体感染的细胞一般选择内细 胞团细胞，因为这类细胞能发育成新个体的各种组织器官（具有发育的全能性），处于囊胚期。
高三生物基因工程练习题及答案 第一节：选择题 1. 现代基因工程技术的突破是建立在下列发现的基础之上的： A. DNA双螺旋结构 B. 基因重组的概念 C. DNA复制的机制 D. 基因的表达调控机制 正确答案：A. DNA双螺旋结构 2. 下列哪个是基因工程的主要应用领域： A. 农业领域 B. 医学领域 C. 环保领域 D. 以上都是 正确答案：D. 以上都是 3. 基因工程按操作方式可分为以下几类，下面哪个选项顺序正确： A. 提取、分离、重组、转移 B. 分离、转移、提取、重组
C. 提取、分离、转移、重组 D. 分离、提取、转移、重组 正确答案：C. 提取、分离、转移、重组 4. 以下哪项不属于基因工程的基本技术之一： A. PCR B. SDS-PAGE C. DNA测序 D. 基因克隆 正确答案：B. SDS-PAGE 5. 哪个技术代表了原创性的DNA测序方法，为测序领域的巨大进步奠定了基础： A. Sanger测序法 B. PCR法 C. 杂交技术 D. 聚合酶链式反应技术 正确答案：A. Sanger测序法 第二节：填空题
1. 基因工程主要通过重组DNA来完成基因的操纵和转移。其中，常用的DNA序列寻找方法是______。 答案：PCR （聚合酶链式反应） 2. 基因工程筛选转化细胞常用的方法是______。 答案：筛选抗生素（抗生素筛选法） 3. 基因工程中，将外源基因与载体进行重组，得到重组DNA，这个过程称为______。 答案：基因克隆 4. 基因工程常用的工具酶是______。 答案：限制性内切酶 5. 将基因工程所重组得到的DNA片段转入细胞内部的过程称为 ______。 答案：转化（也可接受转染） 第三节：解答题 1. 请描述一下基因工程中的PCR技术的原理和应用。 答案：PCR（聚合酶链式反应）技术是一种体外人工合成DNA片段的技术。其原理是通过DNA解旋、引物延伸和DNA复制等步骤，使得DNA在体外被无限制地复制增加。PCR技术能够在短时间内扩增特定的DNA序列，具有高度灵敏性和特异性。在基因工程中，PCR技
基因工程习题集 一、名词解释：（20×4/） 1、基因 2、克隆 3、基因定位 4、PCR 5、基因工程工具酶 6、组织培养 7、限制性核酸内切酶 8、受体细胞 9、生物技术 10、基因沉默 11、重组DNA技术12、逆转录酶13、单克隆抗体14、核移植技术 15、细胞融合16、胞内酶17、凝胶过滤18、固定化酶 19、蛋白质工程 20、生物反应器 二、单项选择题：(60×2/) 1．下列关于基因工程应用的叙述，正确的是（） A．基因治疗就是把缺陷基因诱变成正常基因B．基因诊断的基本原理是DNA分子杂交C．一种基因探针能检测水体中的各种病毒D．原核基因不能用来进行真核生物的遗传改良2．在已知某小片段基因碱基序列的情况下，获得该基因的最佳方法是（）A．用mRNA为模板逆转录合成DNA B．以4种脱氧核苷酸为原料人工合成 C．将供体DNA片段转入受体细胞中，再进一步筛选 D．由蛋白质的氨基酸序列推测mRNA 3．我国科学家运用基因工程技术，将苏云金芽孢杆菌的抗虫基因导人棉花细胞并成功表达，培育出了抗虫棉。下列叙述不．正确的是（） A．基因非编码区对于抗虫基因在棉花细胞中的表达不可缺少 B．重组DNA分子中增加一个碱基对，不一定导致毒蛋白的毒性丧失 C．抗虫棉的抗虫基因可通过花粉传递至近缘作物，从而造成基因污染 D．转基因棉花是否具有抗虫特性是通过检测棉花对抗生素抗性来确定的 4．采用基因工程技术将人凝血因子基因导入山羊受精卵，培育出转基因羊。但是，人凝血因子只存在于该转基因羊的乳汁中。以下有关叙述，正确的是（） A．人体细胞中凝血因子基因编码区的碱基对数目，等于凝血因子氨基酸数目的3倍B．可用显微注射技术将含有人凝血因子基因的重组DNA分子导入羊的受精卵 C．在该转基因羊中，人凝血因子基因存在于乳腺细胞，而不存在于其他体细胞中 D．人凝血因子基因开始转录后，DNA连接酶以DNA分子的一条链为模板合成mRNA 5.科学家通过基因工程的方法，能使马铃薯块茎含有人奶主要蛋白。以下有关该基因工程的叙述，错误的是（） A.采用反转录的方法得到的目的基因有内含子 B.基因非编码区对于目的基因在块茎中的表达是不可缺少的 C.马铃薯的叶肉细胞可作为受体细胞
专题1 《基因工程》单元测试题 一、单选题(每小题只有一个正确答案) 1.在基因工程中用来修饰改造生物基因的工具是( ) A．限制酶和DNA连接酶 B．限制酶和水解酶 C．限制酶和载体 D． DNA连接酶和载体 2.下列说法正确的是( ) A．质粒是广泛存在于细菌细胞中的一种颗粒状细胞器 B．用适当的化学物质处理受体细菌表面，将重组DNA导入受体细菌 C．质粒是基因工程中惟一用作运载目的基因的载体 D．利用载体在宿主细胞内对目的基因进行大量复制的过程不能称为“克隆” 3.某研究小组为了研制预防禽流感病毒的疫苗，开展了前期研究工作。其简要的操作流程如下图。 下列有关叙述错误的是( ) A．步骤①所代表的过程是反转录 B．步骤②需使用限制性核酸内切酶和DNA连接酶 C．步骤③可用CaCl2处理大肠杆菌，使其从感受态恢复到常态 D．检验Q蛋白的免疫反应特性，可用Q蛋白与患禽流感康复的鸡的血清进行抗原—抗体特异性 反应试验 4.与“限制性内切酶”作用部位完全相同的酶是( ) A．反转录酶 B． RNA聚合酶 C． DNA连接酶 D．解旋酶 5.转基因抗虫棉的研制过程中，为了检测抗虫基因是否成功导入，最简捷有效的方法是( ) A．用DNA探针检测受体细胞中的目的基因是否存在 B．用DNA探针检测受体细胞中的目的基因是否转录出相应的mRNA C．直接将培育出的棉株叶片饲喂原本的棉花害虫 D．检测标记基因是否正常地表达出来
6.下列哪项不是表达载体所必需的组成( ) A．目的基因 B．启动子 C．终止子 D．抗青霉素基因 7.下列说法正确的是( ) A．限制性核酸内切酶的识别序列是GAATTC，只能在G和A之间切断DNA B． DNA连接酶能够将任意2个DNA片段连接在一起 C．质粒是能够自主复制的小型双链环状DNA分子 D．基因工程的载体只有质粒一种 8.应用生物工程技术培育人们需要的生物新品种或新产品，提高了经济效益。下图表示培育生物新品种的过程，请据图判断下列叙述中不正确的是( ) A．图中①～⑤过程中都发生了碱基互补配对现象 B．图中①过程需要的工具酶是限制酶和DNA连接酶 C．将PrG导入细胞Ⅱ，则Ⅱ最可能是浆B细胞 D．图中⑤过程需要的培养基中一定含有植物激素和无机养料 9.在已知序列信息的情况下，获取目的基因的最方便方法是( ) A．化学合成法 B．基因组文库法 C． cDNA文库法 D．多聚酶链式反应 10.中国新闻网报道，阿根廷科学家近日培育出了世界上第一头携带有两个人类基因的牛，因此有望生产出和人类母乳极其类似的奶制品。下列叙述正确的是( ) ①该技术将导致定向变异 ②DNA连接酶将目的基因与载体黏性末端的碱基对连接起来 ③蛋白质中的氨基酸序列可为合成目的基因提供资料
基因工程原理练习题及其答案 一、填空题 1．基因工程是_________年代发展起来的遗传学的一个分支学科。 2．基因工程的两个基本特点是：(1)____________，(2)___________。 3．基因克隆中三个基本要点是：___________；_________和__________。 4．通过比较用不同组合的限制性内切核酸酶处理某一特定基因区域所得到的不同大小的片段，可以构建显示该区域各限制性内切核酸酶切点相互位置的___________。 5．限制性内切核酸酶是按属名和种名相结合的原则命名的，第一个大写字母取自_______，第二、三两个字母取自_________，第四个字母则用___________表示。 6．部分酶切可采取的措施有：(1)____________(2)___________ (3)___________等。 7．第一个分离的限制性内切核酸酶是___________；而第一个用于构建重组体的限制性内切核酸酶是_____________。8．限制性内切核酸酶BsuRI和HaeⅢ的来源不同，但识别的序列都是_________，它们属于_____________。 9．DNA聚合酶I的Klenow大片段是用_____________切割DNA聚合酶I得到的分子量为76kDa的大片段，具有两种酶活性：(1)____________；(2)________________的活性。 10．为了防止DNA的自身环化，可用_____________去双链DNA__________________。 11．EDTA是____________离子螯合剂。 12．测序酶是修饰了的T7 DNA聚合酶，它只有_____________酶的活性，而没有_______酶的活性。 13．切口移位(nick translation)法标记DNA的基本原理在于利用_________的_______和______的作用。 14．欲将某一具有突出单链末端的双链DNA分子转变成平末端的双链形式，通常可采用_________或_______________。15．反转录酶除了催化DNA的合成外，还具有____________的作用，可以将DNA- RNA杂种双链中的___________水解掉。 16．基因工程中有3种主要类型的载体：_______________、_____________、______________。 17．就克隆一个基因(DNA片段)来说，最简单的质粒载体也必需包括三个部分：_______________、_____________、______________。另外，一个理想的质粒载体必须具有低分子量。 18．一个带有质粒的细菌在有EB的培养液中培养一段时间后，一部分细胞中已测 不出质粒，这种现象叫。 19．pBR322是一种改造型的质粒，它的复制子来源于，它的四环素抗性基因来自于，它的氨苄青霉素抗性基因来自于。 20．Y AC的最大容载能力是，BAC载体的最大容载能力是。 21．pSCl01是一种复制的质粒。 22．pUCl8质粒是目前使用较为广泛的载体。pUC系列的载体是通过 和两种质粒改造而来。它的复制子来自，Amp 抗性基因则是来自。 23．噬菌体之所以被选为基因工程载体，主要有两方面的原因：一是；二是。 24．野生型的M13不适合用作基因工程载体，主要原因是 和。 25．黏粒(cosmid)是质粒—噬菌体杂合载体，它的复制子来自、COS位点序列来自，最大的克隆片段达到kb。 26．野生型的λ噬菌体DNA不宜作为基因工程载体，原因是：(1) (2) (3) 。 27．噬菌粒是由质粒和噬菌体DNA共同构成的，其中来自质粒的主要结构是，而来自噬菌体的主要结构是。 28．λ噬菌体载体由于受到包装的限制，插入外源DNA片段后，总的长度应在噬菌体基 因组的的范围内。 29．在分离DNA时要使用金属离子螯合剂，如EDTA和柠檬酸钠等，其目的是 。 30．用乙醇沉淀DNA时，通常要在DNA溶液中加人单价的阳离子，如NaCl和NaAc， 其目的是。 31．引物在基因工程中至少有4个方面的用途：(1) (2) (3) (4) 。 32．Clark发现用Taq DNA聚合酶得到的PCR反应产物不是平末端，而是有一个突出 碱基末端的双链DNA分子。根据这一发现设计了克隆PCR产物的。 33．在cDNA的合成中要用到S1核酸酶，其作用是切除在 。 34．乙醇沉淀DNA的原理是。 35．假定克隆一个编码某种蛋白质的基因，必须考虑其表达的三个基本条件：
基因工程期末试题及答案 一、选择题（每题2分，共20分） 1. 基因工程是指利用现代生物技术手段对DNA进行操作和控制的一种技术。 2. 基因工程的主要方法包括基因克隆、PCR、DNA测序等。 3. DNA测序是通过测定DNA序列来确定一个基因的具体结构。 4. 在基因工程中，限制性内切酶用于切割DNA分子，产生特定片段。 5. 基因工程可以产生转基因生物，通过对生物基因进行改造，使其具有特定的特性。 6. CRISPR/Cas9是一种常用的基因编辑工具，可以精确地修改基因组中的特定片段。 7. 基因工程在医学领域可以用于基因治疗，通过修复或替换异常基因，治疗遗传性疾病。 8. 在农业领域，基因工程可以用于改良作物，提高产量和抗病虫害能力。 9. PCR是一种常用的基因扩增技术，可以通过复制DNA片段来放大特定基因。 10. DNA梯度离心是基因工程中常用的分离DNA片段的方法。
二、问答题（每题10分，共30分） 1. 请简要介绍基因工程的基本原理和主要方法。 基因工程的基本原理是通过对DNA进行操作和控制来改变生物的遗传性状。其主要方法包括基因克隆、PCR、DNA测序等。基因克隆是通过将特定DNA片段放入载体中，然后转化到宿主细胞中，使重组DNA在宿主细胞中大量复制。PCR是一种基因扩增技术，通过在DNA复制过程中加入特定引物，使特定基因片段在体外大量扩增。DNA测序是通过测定DNA序列来确定一个基因的具体结构。 2. 什么是转基因生物？请举例说明。 转基因生物是指经过基因工程改造，将外源基因导入到目标生物体中，使其具有新的遗传性状的生物。例如，将耐旱基因导入水稻，使其具有抗旱能力；将Bt毒素基因导入棉花，使其具有抗虫能力。 3. 基因工程在医学领域有哪些应用？举例说明。 基因工程在医学领域可以用于基因治疗、疫苗研发等。例如，利用基因工程技术可以修复或替换患者体内异常基因，治疗遗传性疾病。另外，基因工程也可以用于疫苗研发，通过基因工程技术将病原体的基因导入宿主细胞，产生病原体的抗原蛋白，从而刺激免疫系统产生抗体，用于预防传染病。 4. 基因工程在农业领域有哪些应用？举例说明。 基因工程在农业领域可以用于改良作物、提高产量和抗病虫害能力等。例如，通过基因工程技术将耐盐碱基因导入水稻，使其适应盐碱
基因工程习题及参考答案01 绪论部分 一、填空题 1．基因工程是年代发展起来遗传学一个分支学科。基因工程技术诞生, 使人 们从简单地利用现存生物资源进行诸如发酵、酿酒、制醋和酱油等传统生物技术时代, 走向时代。 2．伴随基因工程技术诞生和发展, 人类能够经过、和等三种关键生产方法, 大量取得过去只能从组织中提取珍稀蛋白, 用于研究或治病。3．Cohen 等在年构建了第一个有功效重组DNA 分子。 4．基因工程两个基础特点是: (1) , (2) 。 5．基因克隆中三个基础关键点是: ; 和。6．年, 美国斯坦福大学等在上发表了题为: “将新遗传信息插入SV40 病毒DNA 生物化学方法: 含有λ噬菌体基因和E．coli 半乳糖操纵子环状SV40 DNA”论文, 标志着基因工程技术诞生。这一工作含有划时代意义, 不过她们并没有。 7．克隆基因关键目有四: (1) ; (2) ; (3) ; (4) 。 二、选择题(单选或多选) ( )1．因研究重组DNA 技术而取得诺贝尔奖科学家是 (a)A．Komberg (b)W．Gilbert (c) P．Berg (d)B．McClintock ( )2．第一个作为重组DNA 载体质粒是( ) （a）pBR322 (b)ColEl (c)pSCl01 (d)pUCl8 ( )3．第一个用于构建重组体限制性内切核酸酶是( ) (a)EcoRI (b)EcoB (c)EcoC (d)EcoRⅡ ( )4．P Berg 构建SV40 二聚体时用了多个不一样酶, 其中( )作用是制造隐蔽5’端。 (a)末端转移酶(b)λ外切核酸酶(c)外切酶Ⅲ(d)DNA 连接酶
高中生物《基因工程》练习题 题号一二总分 得分 一、单选题（本大题共20小题，共20.0分） 1.如图为DNA分子的某一片段，其中①②③分别表示某种酶的作用部位，则相应的酶依次为（） A. 解旋酶、限制酶、DNA连接酶 B. 限制酶、解旋酶、DNA连接酶 C. 限制酶、DNA连接酶、解旋酶 D. DNA连接酶、限制酶、解旋酶 2.下列有关基因工程技术的叙述，正确的是（） A. 重组DNA技术所用的工具酶是限制酶、连接酶和运载体 B. 所有的限制酶都只能识别同一种特定的核苷酸序列 C. 选用细菌为重组质粒受体细胞是因为质粒易进入细菌细胞且繁殖快 D. 只要目的基因进入受体细胞就能成功实现表达 3.如图为基因表达载体的模式图。下列有关基因工程的说法错误的 是（） A. 基因工程的核心步骤是基因表达载体的构建 B. 任何基因表达载体的构建都是一样的，没有差别 C. 图中启动子和终止子不同与起始密码子和终止密码子 D. 抗生素抗性基因的作用是作为标记基因，用于鉴别受体细胞中是否导入了载体 4.一些细菌能借助限制性核酸内切酶抵御外来入侵者，而其自身的基因组DNA经预先修饰能躲避 限制酶的降解。下列在动物体内发生的过程中，与上述细菌行为相似的是（） A. 巨噬细胞内溶酶体杀灭病原体 B. T细胞受抗原刺激分泌淋巴因子
C. 组织液中抗体与抗原的特异性结合 D. 疫苗诱导机体产生对病原体的免疫 5.某目的基因两侧的DNA序列所含的限制性核酸内切酶位点如图所示，最好应选用下列哪种质粒 作为载体（） A. B. C. D. 6.下图是研究人员利用供体生物DNA中无限增殖调控基因制备单克隆抗体的思路流程。下列相关 叙述正确的是（） A. 酶a、酶b作用位点分别为氢键和磷酸二酯键 B. Ⅰ是经免疫的记忆细胞与骨髓瘤细胞融合的杂交瘤细胞 C. 筛选出既能无限增殖又能产生专一抗体的Ⅱ必须通过分子检测 D. 上述制备单克隆抗体的方法涉及转基因技术和动物细胞核移植技术 7.下列关于基因工程技术的说法，正确的是（） A. 切割质粒的限制酶均只能特异性地识别3-6个核苷酸序列 B. PCR反应中两种引物的碱基间应互补以保证与模板链的正常结合 C. 载体质粒通常采用抗生素抗性基因作为标记基因 D. 目的基因必须位于重组质粒的启动子和终止子之间才能进行复制 8.在其他条件具备的情况下，在试管中进入物质X和物质Z，可得到相应产物Y．下列叙述正确 的是（）
《基因工程》 一、选择题（每小题1.5分，共15分） 1．基因工程的创始人是 ( )。 A A. Kornberg B W. Gilbert C P. Berg D S. Cohen 2．关于宿主控制的限制修饰现象的本质，下列描述中只有( )不太恰当。 A 由作用于同一DNA序列的两种酶构成 B 这一系统中的核酸酶都是Ⅱ类限制性内切核酸酶 C 这一系统中的修饰酶主要是通过甲基化作用对DNA进行修饰 D 不同的宿主系统具有不同的限制-修饰系统 3．在DNA的3’-5’链上，基因的起始密码子是 ( ) A ATG（或GTG） B AGT C TAG D TGA 4．II限制性内切核酸酶可以特异性地识别 ( )。 A 双链DNA的特定碱基对 B 双链DNA的特定碱基序列 C 特定的三联密码 D 以上都正确 5．末端转移酶是合成酶类，具有( )活性。 A 3’→5’DNA聚合酶 B 5’→3’DNA聚合酶 C 5’→3’DNA内切酶 D 5’→3’DNA外切酶 6．下面关于松弛型质粒(relaxed plasmid)性质的描述中，( )是不正确的。 A 质粒的复制只受本身的遗传结构的控制，因而有较多的拷贝数。 B 可以在氯霉素作用下进行扩增。 C 通常带有抗药性标记。 D 同严紧型质粒融合后，杂合质粒优先使用松弛型质粒的复制子。 7．关于cDNA的最正确的说法是( )。 A 同mRNA互补的单链DNA B 同mRNA互补的双链DNA C 以mRNA为模板合成的双链DNA D 以上都正确 8．关于T4 DNA Ligase，下列说法中哪一项不正确? ( ) A 是最常用的DNA连接酶。 B 不但能连接粘性末端，还能连接齐平末端。 C 不但能连接粘性末端。 D 最适温度37 ℃。 9．下列关于建立cDNA文库的叙述中，( )是错误的? A 从特定组织或细胞中提取DNA或RNA B 用反转录酶合成mRNA的对应单链DNA C 以新合成的单链DNA为模板合成双链DNA D 新合成的双链DNA克隆到载体上，并导入受体细胞 10．用下列方法进行重组体的筛选，只有( )说明外源基因进行了表达。 A Southem blot B Northem blot C Western印迹 D 原位菌落杂交 二、填空题（每空1分，共25分） 1．限制性内切核酸酶分为三类，基因工程中应用的是____________ _。 2．为了防止DNA的自身环化，可用_____________去双链DNA__________________。 3．切口平移(nick translation)法标记DNA探针时应用的酶是_____ ___ __。 4．基因工程中的3种主要类型的载体是_______________、_____________、__________。 5．野生型的M13不适合用作基因工程载体，主要原因是、和。 6．黏粒(cosmid)是杂合载体。 7．引物在基因工程中至少有4个方面的用途：(1) (2) 、 (3) (4) 。 8．Northern印迹和Southern印迹有两点根本的区别：(1)_ __、，(2)________ 9．PCR反应中常用的酶是______ _ 。
2014年 专题1 基因工程 （天津卷）4.为达到相应目的，必须 ．．通过分子检测的是 A。携带链霉素抗性基因受体菌的筛选 B。产生抗人白细胞介素－8抗体的杂交瘤细胞的筛选 C.转基因抗虫棉植株抗虫效果的鉴定 D。21三体综合征的诊断 【答案】B 【解析】可通过将受体菌接种在含链霉素的培养基中筛选携带链霉素抗性基因的受体菌,A错误；抗人白细胞介素的杂交瘤细胞应通过抗原—抗体杂交技术筛选产生,B正确；在棉花田中人工放入害虫可检验转基因抗虫棉的抗虫效果，C错误;可利用显微镜检测21三体综合征，D错误。 （广东卷）25利用基因工程技术生产羧酸酯酶（CarE）制剂的流程如图14所示，下列叙述正确的是( ） A、过程①需使用逆转录酶 B、过程②需使用解旋酶和PCR获取目的基因 C、过程③使用的感受态细胞可用NaCl溶液制备 D、过程④可利用DNA分子杂交鉴定目的基因是否已导入受体细胞【答案】AD 【解析】过程①是以RNA为模板合成DNA的过程，即逆转录过程,需要逆转录酶的催化，故A 正确；过程②表示利用PCR扩增目的基因，在PCR过程中，不需要解旋酶，是通过控制温度来达到解旋的目的,故B 错；利用氯化钙处理大肠杆菌，使之成为感受态细胞，故C错;检测目的基因是否成功导入受体细胞的染色体DNA 中，可以采用DNA分子杂交技术，故D正确 （课标Ⅱ卷）40．[生物—-选修3：现代生物科技专题］(15分） 植物甲具有极强的耐旱性，其耐旱性与某个基因有关。若从该植物中获得该耐旱基因,并将其转移到耐旱性低的植物乙中，有可能提高后者的耐旱性。 回答下列问题： (1)理论上，基因组文库含有生物的基因;而cDNA文库中含有生物的基因. （2）若要从植物甲中获得耐旱基因，可首先建立该植物的基因组文库,再从中出所需的耐旱基因. (3）将耐旱基因导入农杆菌,并通过农杆菌转化法将其导入植物的体细胞中，经过一系列的过程得到再生植株。要确认该耐旱基因是否在再生植株中正确表达,应检测此再生植株中该基因的 ,如果检测结果呈阳性,再在田间试验中检测植株的是否得到提高。 （4)假如用得到的二倍体转基因耐旱植株自交，子代中耐旱与不耐旱植株的数量比为3∶1时,则可推测该耐旱基因整合到了（填“同源染色体的一条上"或“同源染色体的两条上”）. 【答案】(1）全部部分（2）筛选（3）乙表达产物耐旱性（4)同源染色体的一条上 【解析】（1）基因文库包括基因组文库和cDNA文库，基因组文库包含生物基因组的所全部基因，cDNA文库是以mRNA 反转录后构建的,只含有已经表达的基因（并不是所有基因都会表达)，即部分基因。 （2）从基因文库中获取目的基因需要进行筛选。 (3)要提高植物乙的耐旱性，需要要利用农杆菌转化法将耐旱基因导入植物乙的体细胞中。要检测目的基因(耐旱基因)是否表达应该用抗原抗体杂交检测目的基因(耐旱基因）的表达产物(即耐旱的相关蛋白质);个体水平检测可以通过田间实验，观察检测其耐旱性情况。 （4）如果耐旱基因整合到同源染色体的两条上，则子代将全部表现耐旱，不会出现性状分离。[或“如果耐旱基因整合到同源染色体的一条上，则转基因植株的基因型可以用A_表示(A表示耐旱基因，_表示另一条染色体上没有相应的基因）,A_自交后代基因型为AA∶A_∶_ _=1∶2∶1,所以耐旱∶不耐旱=3∶1，与题意相符 （天津卷）8.(12分）嗜热土壤芽胞杆菌产生的β-葡萄糖苷酶（BglB)是一种耐热纤维素酶，为使其在工业生产中更好地应用,开展了以下试验： Ⅰ.利用大肠杆菌表达BglB酶 （1）PCR扩增bglB基因时，选用基因组DNA作模板。 （2）右图为质粒限制酶酶切图谱。bglB基因不含图中限制酶识别序列。为使PCR扩增的bglB基因重组进该质粒,扩增的bglB基因两端需分别引入和不同限制酶的识别序列。 （3）大肠杆菌不能降解纤维素，但转入上述建构好的表达载体后则获得了降解纤维素的能力，这是因为。 Ⅱ.温度对BglB酶活性的影响 （4）据图1、2可知，80℃保温30分钟后,BglB酶会 ;为高效利用BglB酶降解纤维素，反应温度最好控制在（单选）。 A。50℃ B.60℃ C。70℃ D.80℃