Cloning And Analysis Of NBS-LRR Gene In Peanut

Peanut (Arachis hypogaea L.) is one of the major oil and economical crops in the world and also the highest yield of oil crops in China with the total output accounting for45%of the world’s peanut production. The genetic and breeding research has been paid wide attention. It has made remarkable achievements depends on conventional germplasm resources and breeding of hybrid systems method for processing type, exporting type, oil type, nutrition type, resistance type varieties. But the conventional breeding is short for of long breeding cycle, high cost which resitrict the peanut breeding progress. With further development of molecular biology, molecular breeding gradually reveals its superiority. The application of genetic engineering to transfer exogenous gene into peanut genome, is an effective method to improve the quality of peanut, and has promising further incubation of new peanut variety with high disease resistance.In this paper, several overgos was designed according to the conserved region of NBS-LRR gene family. We obtained full sequences through RACE-PCR (rapid-amplification of cDNA ends). For further study of its function we constructed the expression vector of NBS-LRR gene family and cultivate transgenic tobacco and peanuts. Through the analysis of RT-PCR we study the relationship of NBS-LRR gene between disease resistance and drought resistance.The RACE-PCR analysis results show that according to the conservative region of NBS-LRR gene to design primers for PCR amplification, we cloned the middle segment and full length of two genes named PnAGl-1and PnAGl-2. PnAG1-2was identified as1344bp, with open reading frame of1098bp, encoding365amino acids. The full-length of PnAGl-1are1088bp, with an open reading frame of741bp, encoding247amino acids. Among them the Medicago truncatula Rps-k-2gene has the highest similarity. The similarity of PnAGl-1and PnAGl-2were85%and87%respectively. Similarity with At3g14460gene in soybean is83%and82%.The NBS-LRR gene cloning results showed the gene sequences of PnAG1and PnAG3in peanut as well as the corresponding RNA interference sequence have correctly cloned. Restriction enzyme digestion and sequencing results showed the sequence was correctly ligated to the PUC19vector and PRi101-AN plant expression vector.Transgenic results showed that transgenic plants of PnAG1and PnAG3was obtained, which shows that the vector has been successfully implanted in the genome of tobacco.Real-Time PCR analysis showed that the expression of PnAG3, PnAG1and PnAG1-1the expression in the seedling stage is lower than florescence in leaves; PnAGl-2showed the opposite.PnAG1-2is active in seedling development process while the other three genes in flowering development are more active.The expression of PnAGl and PnAG3, PnAGl-1in seedling stages are higher than florescence; PnAGl-2showed the opposite. While PnAGl and PnAG3play a very important role in the resistance to Aspergillus flavusing drought condition. According to the results of PnAGl and PnAG3high expressison during Aspergi Hums flatus effection, we also know the expression level depends on drought, too. It shows that the relationship between resistance and drought resistance is closely related.In this paper, we use tobacco as transgenic object of peanut gene to study its function, and study the potential relationship of gene function between drought stress and disease resistance using fluorescence quantitative methods. This laid the foundation of explaining the relationship between drought and resistance. This paper provide an important basis for the study of molecular breeding and gene function research methods of analysis.