Cloning And Characterization Of Gene Resistant To Aspergillus Flavus And Their Transformation Into Peanut

Peanut is an important oilseed crops and cash crops in our country.Peanut aflatoxin contamination caused by Aspergillus flavus's infection is the main factor affecting peanut production,processing and trade.Up to now,there has not been any varieties and germplasm developed showing stable and high resistance to A.flavus.In recent year,we suggested that A.flavus contamination could be effectively solved employing modern gene engineering technology.In the paper,we isolated momordica charantia ribosome inactivating protein gene(RIP),tobacco chitinase gene(CHI),tobaccoβ-1,3-glucanase gene(GLU)and the rabbit defensin-2 gene (NP2)sequences from available vectors in the laboratory by PCR and obtained defensin gene 1(NP1)from rabbit by homology cloning.Then evaluated the resistance to Aspergillus flavus by prokaryotic expression and in vitro inhibition tests. Based on it,constructed plant expression vectors with CHI,GLU,and a peanut's trypsin inhibitor gene(TI),which were used to transform peanut afterwards.The results are as follows1.NP1 gene was cloned from the rabbit pancreas' RNA by RT-PCR using specific primers devised according to the published sequence of NP1 on GenBank. Sequencing result showed the obtained sequence was 401 bps in length with 99% identity with the interested sequence on web.2.Prokaryotic expression vectors,pGEX-RIP,pGEX-NP1,pGEX-CHI, pGEX-GLU and pGEX-NP2,containing RIP,NP1,CHI,GLU and NP2,respectively, were constructed with pGEX-4T-1 vector,and then were introduced into a host bacterium BL21.Induced expression products were isolated,and assayed by SDS-PAGE electrophoresis,indicating that the fussion protein formed specific bands at about 59kDa,37 kDa,31kDa,61kDaand 64 kDa,respectively.In vitro inhibiting A.flavus strain's growth were cheched and protein products of CHI gene and GLU gene may show higher inhibiting the growth of Aspergillus flavus,but peanut trypsin inhibitor Ti and Np1 may be weaker.(3)Three plant expression vectors with GLU,CHI,and TI genes were constructed and named pSC1300-GLU,pSC1300-CHI,pSC1300-TI.They were transformed into peanut together with a premier constructed vector pSC1300-NP1.The resulting sistant seedlings were sampled and detected by PCR,showing that NP1 gene has inserted into peanut genome.Above all,this study aimed at taking advantage of the anti-fungal properties, transformating resistance genes into peanuts through molecular breeding methods, The result obtained could supply with foundation for employing genetic engineering means to solve A.flavus contamination...