Application of Modern Biotechnology in Rape Breeding (2)
Dragon Pearl,Mo Li Long Zhu,Jasmine Tea Prettyfly Technology Co., Ltd. , http://www.prettyflytea.com
2.1 Herbicide Resistance The introduction of herbicide tolerance into crops is a new way to increase the selectivity and completeness of herbicides. In the rapeseed genetic engineering, the introduction of an anti-glyphosate-induced mutant gene of EPSP synthase (5-Enolpyruvylshikimate-3-phosphate synthase) was effective. Glyphosate is a non-selective broad-spectrum herbicide that blocks the synthesis of aromatic amino acids by inhibiting the activity of EPSP synthetase and eventually leads to the death of the test plants. A mutant strain has been isolated from EColi, which contains a glyphosate-resistant mutant gene of EPSP synthase, which is introduced into crops. When glyphosate is used, the crop is not damaged. Because glyphosate is non-toxic, has no residue, is easily decomposed, and does not pollute the environment, people attach great importance to the genetic manipulation of the glyphosate-resistant EPSP synthase gene. At present, there are two Glyphosate-resistant transgenic rape lines in Canada, and a joint Canadian rape variety test from 1992 to 1994. These strains are similar in yield to current varieties, but their quality and resistance are enhanced. 2.2 Insect-resistant culture Insect-resistant plants are an important application area of ​​genetic engineering, not only for improving crops, but also for the seed industry and agrochemicals that cannot be underestimated. In terms of insect resistance, it is mainly coded by cloning. The toxin protein gene (also called the insecticidal crystal protein gene) of Bocillas thuringiensis (BT) is transferred to plant cells to obtain insect-resistant transgenic plants. It has now been transferred to tobacco, tomatoes and cotton. In the Jiangyan insect-resistant genetic engineering, the Bacillus thuringiensis toxin protein gene has been transferred into rapeseed, broccoli, broccoli, and cabbage. Using some protease inhibitor genes, insect resistant plants can also be obtained. For example, the British has overcome the cowpea trypsin inhibitor gene, the gene is transferred to the plant, it produces inhibitors that can destroy the body trypsin activity. After pests feed the transgenic plants, they die due to indigestion. 2.3 Disease-resistance virus The harm to plants is one of the greatest losses in agricultural production. At present, it is widely used to control and avoid killing insects, breed breeding disease-resistant genes, produce virus-free vaccines and inoculate The morbid attenuated strains have more or less restrictive factors, such as the effect of achieving cross-protection, resulting in poor results or adverse effects. There are several ways to use plant engineering to prevent viruses. 1 Introduction of the viral coat protein gene: the use of the introduced coat protein gene to form cross-protection to prevent or reduce the risk of viruses. The anti-viral transgenic vegetable crops that have been obtained include tomatoes, potatoes, and peppers. In the United States in 1986, tomato plants transformed with the TMV coat protein gene were obtained. Under field test conditions, only 5% of the plants infected with TMV coat protein were infected with TMV, and the yield was not reduced, while the incidence of control plants was 99%. Reduce production by 26% to 35%. Virus coat protein genes that have been successfully transformed include potato virus X (PVX), potato virus Y (PVY), cucumber mosaic virus (CMV), and soybean mosaic virus (SMV). 2 Introduction of viral RNA satellite RNA cRNA: In 1986 British scientists converted CMV satellite RNA to cDNA. Then it was transferred to the plants and the first time it received engineering plants resistant to CMV. Chinese scholar Zhao Shuzhen also obtained similar transgenic plants. 3 Antisense RNA of the virus: Japanese scientists introduced CMV antisense RNA gene into pepper in 1993 and explored the application value of antisense technology in anti-virus breeding. The mechanism of disease resistance is to reversely bind the genome of the virus to the promoter and transfer it to the plant so that the transgenic plant encodes RNA of the antisense gene. When the exogenous RNA virus invades, the antisense RNA forms a complementary structure with it. Double-stranded structure to prevent virus replication and reduce virus damage. In addition to the above three methods, disease-resistant varieties can also be bred using plant-encoded resistance genes and Other genes on the virus. 2.4 Quality Improvement According to Davies (1992), the enzyme that catalyzes the unsaturation during the fatty acid metabolism is the 18-carbonyl carrier protein dehydrogenase in the plastids. The antisense RNA gene was introduced into rapeseed and phthalocyanine. As a result, the saturated 18-carbon alkanoic acid content in transgenic plants increased from 2% to 40%, a 20-fold increase. However, the oil content is only half that of normal seeds. According to Kuntzon et al., the lauric acid acyl carrier protein thioesterase gene isolated from California laurel was introduced into oilseed rape, so that the content of lauric acid (13 carbon saturated fatty acid) in transgenic rape seed oil was as high as 50%. In addition, Krebbeors et al. (1991), Stayton et al. (1991), Altenbach et al. (1992) reported that Arabidopsis thaliana and pea 2S albumin genes and Brazil nut-rich methionine seed protein genes were introduced through A. tumefaciens seedlings. The total amount of protein in transgenic rape increased exponentially, and the methionine and lysine contents increased significantly. These facts all indicate that it is possible to improve the composition of lipids and proteins in genetically engineered seeds. 2.5 Self-incompatibility transition Self-incompatibility (SI) There are two major systems of sporophyte and gametophyte. The incompatible system of sporophytes is incompatible pollen tube growth stagnation on the stigma surface, gametophyte not The affinity system grows out of pollen tubes due to incompatible pollen, and the stagnant growth of pollen tubes usually occurs after entering the stigma. Brassica oleracea is only a sporophyte system, and its S-locus contains two polymorphic genes. That is, the S-locus glycoprotein (SLG) and S-receptor kinase (SPK) genes, which are isolated, are separated by about 200 kb. A structural region resembling SLG in the SRK gene, a putative transmembrane domain, and a kinase domain. Brassica napus is a self-affinity plant. After the SLG gene is transferred into Brassica oleracea (Brassica oleracea), Brassica napus becomes a self-compatible strain. This may be due to a beneficial suppression of the SLG gene in Brassica oleracea, resulting in changes in the stigma. 2.6 Fertility The TA29 gene that determines plant fertility and the transgenic hybrid rapeseed have made a breakthrough. The TA29 nuclease gene was first discovered by Goldberg RB in a tobacco flower organ. This gene can be expressed in crops such as rapeseed. According to Marlani C et al., when the exogenous TA29 nuclease gene is specifically expressed in the tapetum, the tapetum cells are aborted, and the tapetum cells are mainly for improving the nutrition of the pollen grains, resulting in abortion. Pollen development is abnormal and manifests as male sterility. In order to restore fertility, Marlani C also designed a fusion gene consisting of a tapetum-specific promoter and a TA29 nucleic acid repressor gene to be introduced into a plant and hybridize with the above-mentioned male sterile strain obtained after the introduction of the TA29 nuclease gene in F1. In the generation, TA29 nuclease activity was inhibited due to TA29 nucleic acid repressor gene expression, thereby restoring fertility. To make genetically engineered male sterility safer, Mariani C designed a transformed plant in which the TA29 nuclease gene was put in series with the bar gene (PPT acetyltransferase, which encodes a herbicide-resistant phosphoflavonoid). The offspring produced when this transgenic male sterile plant is crossed with normal rapeseed can be treated with a herbicide, selectively killing the fertile plant while retaining the sterile plant. Now Belgium PGS (1993) has used this set of materials to produce hybrids. 3. Molecular marker Molecular marker refers to an amplified and detectable DNA sequence that is linked to a specific gene or marker. The classical molecular marker RFLP (Restriction Fragment Length Polymorphism) has so far only 10 years of history, but people have used it to construct hundreds of genetic linkage maps of plant molecular markers. Later, various molecular markers based on PCR technology were developed, such as SSR, RAPD, SCAR, AFLP, and so on. These molecular markers have their own merits, and many special articles have been introduced. Here is a summary of the utility of molecular markers in assisted crop breeding. In essence, molecular markers are consistent with the morphological markers and biochemical markers used to construct classical cytogenetic linkage maps. The difference is that compared with the latter two, the former directly reflects the variation in the DNA sequence, and has an infinite number, so it has a wider use in the auxiliary crop breeding. 3.1 DNA Fingerprint Analysis of Crop Variety Resources This analysis will not only lead to the evaluation, classification and use of the nature of genetic resources, but will also play a role in the purity determination of varieties and the protection of intellectual property rights of varieties. 3.2 Marking Important Genes Some important genes, such as detection of resistance genes, are not only time-consuming but are also limited by plant developmental stages. The use of molecular markers closely linked to these genes undoubtedly contributes to the selection of specific genotypes during the breeding process. If the linkage relationship between the molecular marker and the target gene is used to construct a molecular marker genetic linkage map similar to the cytogenetic map, then the molecular marker will also have the following applications: 1 Backcross breeding in secondary backcross breeding needs to be solved One of the problems is the chain of cumbersomeness. The use of molecular markers may detect individuals who have exchanged on both sides of the gene of interest, and thus can reach the goal of backcrossing in conventional backcross breeding only 10 times after only two or three backcrosses. 2 Whole genome selection The entire genome composition of each preselected individual can be analyzed by means of a saturated molecular marker linkage map. On this basis, individuals with more than one target trait and the best genetic basis were selected. 3 Heterosis analysis and prediction Heterosis is derived from the heterozygosity of DNA. For the first time, molecular markers provide a means to accurately determine the heterozygosity of DNA in hybrid combinations, and for the first time it is possible to predict heterosis from the DNA level. Using molecular markers, it is also possible to artificially breed parents that may be highly heterozygous for important fragments of the DNA sequence, thereby formulating a super-dominant F1 combination. 4. Conclusion The modern plant breeding science at the turn of the century is characterized by the close penetration of the three major biotechnologies of tissue culture, molecular cloning and molecular markers and breeding practices. Tissue culture technology has become more and more mature and has become an important auxiliary tool for conventional breeding of rapeseed. With the increasing saturation of genetic maps and RFLP marker maps, based on the establishment of correspondence between PCR marker maps and physical maps, breeders will be in the near future. The use of molecular markers is expected to increase selection efficiency and to produce better varieties faster. In addition to playing a huge role in herbicide resistance, disease resistance, and insect resistance, genetic engineering also has attractive prospects for the creation of new male sterile materials and full use of heterosis.