Molecular Cloning And Analysis Of Expression Levels Of Buchnera GroEL Gene Of Aphis Glycines(Hemiptera: Aphididae) And Acyrthosiphon Solani(Hemiptera: Aphididae)

Aphid is one of the most pernicious insect vectors transmitting plant virus.Many virus disease s in crops and vegetables transmitted by aphid cause severe economic losses. Accordingly, it is of gr eat necessity to fully understand the molecular mechanism underlying aphid transmission and effect ive measures should be taken to restrain aphid-borne virus diseases. Numerous molecular determina nts from plant virus on aphid transmission have been identified in previous studies. However, the ap hid vector factors involved in aphid transmission remain poorly understood. Much attention has bee n paid to Buchnera GroEL of aphid, which is a type of protein involved in virus cireulative transmis sion. This present research aims at the main pests of Aphis glycines and Acyrthosiphon solani, throu gh the design of specific primers, using RT-PCR from Aphis glycines(Aphis glycines Matsumura) a nd Acyrthosiphon solani(Acyrthosiphon solani Matsumura) within the cloned buchnera GroEL gen e, and the prokaryotic expression of genes in vitro related research, in order to make clear the soybe an aphid and tomato without net aphid transmission part of the mechanism of soybean Mosaic virus disease, as well as aphids and soybean Mosaic virus disease prevention and control to provide theo retical guidance and scientific basis, the main results are as follows:(1)From hei long jiang province northeast agricultural university in Harbin xiangfang practice base collection of Aphis glycines and Acyrthosiphon solani, transfer in heated indoor illumination i ncubator breeding propagation. The full length sequence of this gene in the buchnera GroEL of Aph is glycines and Acyrthosiphon solani was amplified and sequenced. Furthermore, a nucleotide sequ ence comparison was made among several homologous genes. The two Buehnera GroEL genes len gth of Aphis glycines and Acyrthosiphon solani are all 1647 bp. Their GenBank aeeession numbers a re KJ543522 and KJ543523. Sequences anaysis indicated the Buchnera GroEL eneode 548 amino a cids. A phylogentic tree was constructed based on amino acids deduecd.(2)The cloned Buchnera GroEL genes of Aphis glycines and Acyrthosiphon solani were ligated into pET-30 b expression vector for construction pETAG and pETAS. They were transformated into E.coli BL21(DE3) for probing into their expression patterns. The SDS-PAGE eleetrophoresis result s suggested that pETAG and pETAS expression vectors can expressed a molecular weight of 69 kD at 37 ℃, 10 ℃, 25 ℃, under the condition of the 200 RMP, concentration of 0.8 mM/ml of IPTG in duction 4 h, and the lower the temperature is, the higher the expression level will be. The results sug gest that GroEL gene from Aphis glycines and Acyrthosiphon solani could be expressed in vitro successfully, which lays a solid foundation for the studies on the mechanism underlying SMV transmis sion by Aphis glycines and Acyrthosiphon solani.(3) By real-time PCR(q RT-PCR), A gene expression comparison between the bunerah GroEL in Aphis glycines and Acyrthosiphon solani was made. The results demonstrate that DNA abundanc e of Aphis glycines in the two kinds of aphids the Buchnera GroEL and Acyrthosiphon solani increa sed significantly with the extension of time of feeding SMV, the expression level of Buchnera GroE L in wingless and winged adults were significantly higher than those at other four nymphs time-poi nts. All these results above-mentioned indicated that Buchnera GroEL gene of Aphis glycines can st ably be expressed and correctly in E. coli, and the expression of Buchnera GroEL was induced in A phis glycines and Acyrthosiphon solani after feeding on SMV, which increases the possibility of SM V widespread in soybean field. However, whether the amino acid change could lead to the changes of protein structure and the expression differences between two kinds of aphids still need to be furth er studied.