Investigation Of Peanut Transformed With Cry8Ea1 And Cry8Ha1 Gene From Bacillus Thuringiensis

Peanut is one of the most important oil crops, and China is a large producer, consumer and exporter of peanuts. It is very difficult to control white grubs through routine chemical and biocontrol methods owing to their special habits, resulting in occurrence of white grubs more and more severe yearly, and yield and quality of peanuts were seriously affected. So new pathway needs to be used for control of white grubs. The cry8-type genes from Bacillus thuringiensis are toxic to a great deal of Coleopteran pests such as leaf beetles, scarabs and so on. cry8Ea1, cry8Ha1 genes which had insecticidal activity against Holotrichia parallela were cloned by our laboratory and with own intellectual property rights. So establishment of effective peanuts transformation system and construction of transgenic peanuts with resistance to insects are important theoretical significance and practical value. In this study, Agrobacterium rhizogenes-mediated transformation system was established firstly.Some factors which affected transformation efficiency were optimized. Then composite plants with cry8-transformed roots were obtained, and the resistance of transformed roots was certified quickly.Consequently, the Agrobacterium tumefaciens mediated transformation of peanuts was explored and peanuts transformed with cry8 gene were obtained. The main results were as follows: Construction of high-expression vectors of peanut. Matrix attachment region was cloned, and ligated into pCAMBIA2300 vector. The basal vector which contains two MAR sequences was constructed. Root-specific promoter of tobacco TobRB7 was cloned. Root-specific promoter TobRB7 and constitutive promoter 35S were introduced into basal vector pDMAR, respectively. Meanwhile,Ωsequence, Kozak sequence, and signal for endoplasmic reticulum retention were fixed around cry8-gene. Consequently, vectors which harbored cry8 genes driven by root-specific promoter TobRB7 or constitutive promoter 35S were constructed, and transformed into Agrobacterium rhizogenes and Agrobacterium tumefaciens respectively.A. rhizogenes-mediated transformation of peanut was established. Vector pGFPGUSplus which harbored reporter genes gfp and GUS were introduced into peanut roots via A. rhizogenes. The results of Southern blots showed that gfp gene was inserted into peanuts genome. RT-PCR assay and GUS staining detection demonstrated that transcription of gfp gene and expression of GUS gene were proceeding normally. The above results proved that exogenous genes could be transferred into peanut root system via this method. Factors, such as plant genotype, A. rhizogenes culture stage, were evaluated. The optimal procedure for A. rhizogenes-mediated induction of peanuts transformed roots lines was as follow: 5-day old seedlings without hypocotyls were microinjected with 5×107 cells from exponential growth phase and placed on MS medium supplemented with 50μmol l-1 acetosyringone for 2 days. The maximal transformation efficiency was 61%. It was an efficient system that it only took 45 days to get abundant roots for molecular analysis.Transgenic root systems were obtained. cry8Ea1 and cry8Ha1 gene driven by constitutive promoter 35S were transferred into Baisha1016, and 21 and 33 composite plants were positive for RT-PCR detection, respectively. Those plants were transferred to pots and exposed to Holotrichia parallela larvae for bioassay. Finally, 62.5% and 66.7% of the composite plants developed strong root system and grew well. The results of bioassay demonstrated that modified vectors could insert exogenous genes into peanut genome and the synthetic cry8Ea1 and cry8Ha1 gene had insecticidal activity against Holotrichia parallela.A. tumefaciens-mediated transformation of peanuts was established. Young leaves and cotyledons were used to establish tissue culture system of peanut, and the maximum efficiency of shoot induction was 40.9% and 48.3% respectively. Some kanamycin-resistant seedlings were obtained through soaking leaves and cotyledons in Agrobacterium tumefaciens. There were 30 and 5 seedlings were positive for PCR analysis. The result of ELISA showed that the percentage of Cry8Ha1 protein in leaves reached 0.24%.A rapid system to assess the performance of cry8 genes in peanut roots was established. Transgenic peanuts with cry8Ea1/cry8Ha1 gene were obtained through Agrobacterium tumefaciens mediated transformation. All of these investigations laid the foundation for developments of peanut varieties with resistance to subterranean insects.