Construction Of Multivalent Plant Express Vector Of Chi,rip And DREB1A Gene And Studies On Genetic Transformation Into Cucumber

Cucumber plays an important role in vegetable supply, but the yield is declined every year because of its fungi disease. One of the common protecting methods is chemical medicine. Although it has some effect, it also brings some disadvantages, such as heavy polluted environment, maladjustment ecology and harm to human healthy. It is testified that culture the fungi-resistant cultivates is the most economic and available way. However, the conventional breeding can't meet the need of cultivating the fungi-resistant varieties, because of its long period, short fungi-resistant genes and disappearing resistance with the changes of pathogen. So, using the genetic engineering to breed the fungi-resistant varieties is a available method. But there are some problems in fungi-resistant engineering of cucumber One is that a single gene only can restrain a few varieties of fungi, and the other is that it is difficult to obtain a large quantity of transgenic plants because of lower rate of transformation. Transcription factor DREB1A is cloned and identified from Arabdopsis thaliana. It can regulate the report gene under drought ,cold and high salt stress and plays an important role in the signal conduction under all above stress. Transcriptional factor DREB1A can control the expression of many genes involved in antibiotic stress. It has much more advantage transferring a transcriptional factor than a single gene in the stress tolerance genetic engineering. The expression of rd29A gene of Arabdopsis thaliana is induced by drought ,cold and high salt stress. There are two DRE cis–acting element involved in responding to these stress signals in its promoter region. So it is an effective inducible expression promoter that can be used in genetic engineering of improving stress tolerance. According to above-mentioned facts, this study took cucumber as material to establish regeneration system and genetic transformation system. One multivalent plant express vectors was constructed, including ribosome inactivating protein (rip) gene,chitinase (chi) gene and DREB1A gene,which were regulated respectively by constitutive expression promoter double CaMV35S,E12 and inducible expression promoter rd29A. Cucumber was transformed by Agrobacterium Tumefaciens with the multivalent plant express vectors and obtained PCR positive plants(T0 and T1). The stress tolerance experiments showed that the drought stress tolerance of T1 transgenic cucumber has been improved because of the expression of DREB1A gene. The research work will provide the function for cultivating transgenic variety that resist to osmotic stress and fungi disease and studying resist mechanism of rip gene,chi gene and DREB1A gene. The main results were summarized as follows: (1) Construction of plant expression vectors One multivalent plant express vectors pBDCR was constructed, including that ribosome inactivating protein (rip) gene,chitinase (chi) gene and DREB1A gene, which were regulated respectively by constitutive expression promoter double CaMV35S,E12 and inducible expression promoter rd29A. (2) Establishment of cucumber regeneration system ①Identified adventitious buds inducing medium of cucumber"JinYanSiHao":MS+1mg/L 6-BA, 30g/L Suc, 0.8%Agar, pH5.8. ②Differentiation frenquency of adventitious buds,the number of buds per explant and efficiency of differentiation are different of different genotypes :"ChangChunMiCi"and"JinYanSiHao".differentiation rate of adventitious buds are 85.8% ,69.7% respectively. The number of buds per explant are3.9 ,3.4 respectively. Efficiency of differentiation are 334.6 %,236.9 % respectively. (3) Establishment of cucumber genetic transformation system mediated by Agrobacterium Tumefaciens ①Identified the concentration of Km on stage of adventitious buds differentiation: Km concentration of"JinYanSiHao"is 25 mg/L. ②Resistant buds of "ChangChunMiCi"were obtained, which were transformed with LBA4404 (pBDCR). The rate of resistant buds are 39.79%; Resistant buds of "JinYanSiHao"were obtained. which were transformed with LBA4404 (pBDCR). The rate of resistant buds are 25.39%; (4) Melecular biology indentification of transgenic plants ①PCR positive plants of "ChangChunMiCi"were obtained, which were transformed with LBA4404 (pBDCR). 41 plants out of 142 detected Kmr plants of "ChangChunMiCi"were positive, The rate of PCR positive are 28.87%. The PCR positive plants number of chi ,rip and DREB1A gene are 57 ,50 ,53 respectively.The rate of PCR positive are 40.14 %,35.21%,37.32% respectively. ②PCR positive plants of "JinYanSiHao"were obtained, which were transformed with LBA4404 (pBDCR). 46 plants out of 151 detected Kmr plants of "JinYanSiHao"were positive, The rate of PCR positive are 30.46%. The PCR positive plants number of chi ,rip and DREB1A gene are 57 ,52 ,65 respectively.The rate of PCR positive are 37.75%,34.44%,43.05% respectively. ③The efficiency of transformation is different of different genotypes. The efficiency of transformation of "ChangChunMiCi"which were transformed with LBA4404 (pBDCR) is 11.49%. The efficiency of transformation of "JinYanSiHao"which were transformed with LBA4404 (pBDCR) is 7.73%. ④PCR positive plants of "ChangChunMiCi"(T1)were obtained, which were transformed with LBA4404 (pBDCR). 75 plants out of 377 detected Kmr plants of "ChangChunMiCi"were positive of DREB1A gene, The rate of PCR positive are 19.89%. 45 plants out of 377 detected Kmr plants of "ChangChunMiCi"were positive of chi,rip gene, The rate of PCR positive are 11.94%. (5) Biology Identification of transgenic plants ①The stress tolerance experiments showed that the drought stress tolerance of T1 transgenic cucumber has been improved because of the expression of DREB1A gene. The result of Pro analysis showed that the Pro amount of T1 transgenic cucumber has been improved. ②The Manicol sensitivity experiment of T0 transgenic seed showed that resistant to drought of seed has been improved ③P.cubensis experiment is being.done.