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Studies on the Transmission Mechanism of Tomato Yellow Leaf Curl Virus by Bemisia Tabaci

Author: ZuoWenZuo
Tutor: WangXiangJing
School: Northeast Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Bemisia tabaci B-biotype Q-biotype Tomato Yellow Leaf Curl Virus (TYLCV) GroEL Real-time qPCR Prokaryotic expression
CLC: S433
Type: Master's thesis
Year: 2012
Downloads: 158
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The tobacco whitefly, Bemisia tabaci (Gennadius), is one of the most invasive and devastating insect pests in the world, colonizes more than600species of plants including vegetables and cotton. B. tabaci B biotype invaded China and caused great economic losses during the nineteen ninties. It was not until2003that B. tabaci Q biotype was firstly found in Yunnan, and displaced B. tabaci B biotype gradually in most areas in China during the recent years. Tomato Yellow Leaf Curl Virus (TYLCV) is an important plant virus transmitted by B. tabaci in a circulative manner via the hemolymph, which have brought great losses to the tomato production from south to north China, exploded along with the spread of B. tabaci Q biotype. However, molecular mechanism of the TYLCV transmission by B. tabaci and TYLCV disease explosion along with the replacement of B biotype by Q biotype B. tabaci are not clear now. In this present study, the important B. tabaci B and Q biotypes were used to compare the TYLCV acquisition of two biotypes B. tabaci in the same acquisition access period using real-time qPCR technique firstly. The virus transmission-related gene, GroEL gene, encoded by the secondary endosymbiont, Hamiltonella, of the in B. tabaci B and Q biotypes were cloned, sequenced and expressed in vitro to clarify the partial mechanisms of TYLCV transmission by B. tabaci, and provide scientific basis for the prevention and management of B. tabaci and TYLCV. The main results are as follows.(1) The biotypes of B. tabaci and their infection ratio of TYLCV from Beijing and Langfang, Hebei were carried out in autumn2011. The result showed that the B. tabaci with the TYLCV infection rate collected from Haidian district and Changping distrct in Beijing reached45%and20%respectively, and those from Langfang, Hebei province were100%. The biotypes of all the detected samples were B. tabaci Q biotype, but not B or other non B/Q biotype. The detection results suggest that B. tabaci B biotype in partial areas in Beijing and Hebei has been totally displaced by Q biotype and TYLCV appeared in the studied area is transmitted by B. tabaci Q biotype. The TYLCV occurrence changes in different locations.(2) The differences of the capacities of the acquisition for TYLCV by two biotypes B. tabaci during the same acquisition access periods were compared with real-time qPCR technique. The result showed that the virus quantity acquired by the B. tabaci Q biotype was significantly higher than that B biotype in the tested5different acquisition access periods (6-12h). What’s more, Q biotype B. tabaci reached the steady level at12h after feeding on the TYLCV-infected tomato plants, showing12h earlier than B biotype. The above results suggest that B. tabaci Q biotype exhibits a stronger ability than B biotype in both the quantities of virus acquisition and the acquisition speed for the TYLCV.(3) The virus transmission-related gene, GroEL, excreted by the secondary endosymbiotic, Hamiltonella, of B and Q biotype B. tabaci were cloned and sequenced. The results showed that the GroEL gene is a kind of highly conservative gene, with1668bp in full length, encoding555amino acids. The GroEL genes from B and Q biotype B. tabaci on the same host had a high sequence similarity of99.94%and the amino acid sequences revealed99.82%homology. There was one point mutation showing263amino acid changes (V→A) from B to Q biotype B. tabaci. The GroEL genes were quantitatively expressed, and the expressions in Q biotype exhibited1.66-fold and1.28-fold significantly higher than B biotype for the virus-infected and non-infected populations (P﹤0.05) respectively. However, whether the amino acid change could lead the changes of protein structure and bring out the expression differences between the two biotypes B. tabaci still need further study.(4) GroEL gene, from the secondary endosymbiont, Hamiltonella, of B and Q biotype B. tabaci, were proved to encode a62kD protein containing555amino acids. The full sequences of the GroEL gene were cloned and ligated into the expression vector pET30a+. The recombinant plasmids were expressed in BL21-DE3competent cells. The69kD fusion proteins were obtained through a Western blot. The results suggest that GroEL gene from two biotype B. tabaci were expressed in vitro successfully, which lays the foundation for the studies on the mechanism of TYLCV transmission by the B. tabaci B and Q biotypes.

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