Molecular Analysis Of The Complete Genome Of SCYLV-HN1 And Construction Of Bait Plasmds For Studing CP And P0 Proteins Interaction With The Host

Sugarcane is one of the most important commercial crop plant in tropical and subtropical. It has served as a source of sugar for hundreds of years, it is accounting for more than 60 percent of global sugar products, and more than 90 percent of Chinese sugar products. At the same time, sugarcane is one of the most important energy crop in the world. Recently, Sugarcane has been used to produce bioethanol, a renewable bio-fuel energy source. Sugarcane yellow leaf syndrome (SCYLS) was detected in the late 1990s first in Hawaii, and was detected in 2001 first in China. Nowadays, the disease has spread to all the world’s sugar cane producing countries. It is a worldwild viral disease which threat to sugarcane production. The incidence rate of SCYLS is between 10%—100%.Sugarcane yellow leaf virus (SCYLV) is a causal agent of sugarcane yellow leaf syndrome. Today analysis revealed that SCYLV belongs to Polervirus which is a member of the Luteoviridae family. Now 16 whole genomic of SCYLV had been reported in Genbank in the world, but there was few report about the fuction of SCYLV protein.The paper aim to clone the complete genome of SCYLV Hainan isolate (SCYLV-HN1); and to study the relationship of SCYLV-HN1 between other SCYLV isolates which have been reported in Genbank, in order to determine the origin of Hainan SCYLV; to constuct the prokaryotic expression plasmid and bacterial two-hybrid bait plasmid of CP and PO. The main conclusions obtained in this paper are as follows:1. SCYLV-HN1 isolate genome sequence was successfully cloned. There are 5837 nt in the genome sequence, contained 6 open reading frames (ORFs). Analysis The homology of SCYLV-HN1 genome sequences with the other islates which have been repoted in the world. The results show that the genome of the Hainan isolate and BRA had high similarity which was up to 98.5% to 99.0%. And the similarities of the Hainan isolate to CHN-YL1(AM072752), CHN-FJ1(GU190159) and CHN-GD (GU327735) were 98.6%,98.5% and 86.7% respectively.2. The prokaryotic expression of CP and PO gene.A specific primers used in RT-PCR is designed by ourselves according to the sequences of SCYLV-CP and SCYLV-PO gene published. A target fragment of CP and PO are isolated by RT-PCR with RNA isolated from diseased leaves as template. The complete SCYLV-CP and SCYLV-PO gene are got according to the sequence analysis. The open reading frame(ORF) of the CP gene is 591 nt and encodes 196 amino acid residues; the PO gene is 771 nt and encodes 256 anmino acid residues. The plasmid of pET32a(+) is taken as vector bone, the prokaryo expression plasmids named as pET32a-CP and pET32a-PO contained the target gene of CP and PO is constructed. The recombinant plasmids pET32a-CP and pET32a-PO were tested by double digestion tests and sequncing, then the recombinant plasmids were transformed into competent cells of E. coli BL21(DE3) pLySs and induced by IPTG to express the target gene. The PO fusion protein was expressed as inclusion body and the CP fusion protein was expressed in high level and mainly as soluable protein. The molecular weight of CP fusion protein expressed by pET32a (+)-CP gene is about 40 kDa, it is similar to the theoretic value of 21.719 kDa according to the ORF of the CP gene and protein of 18 kDa according to the ORF of the pET32a (+) gene. The molecular weight of PO fusion protein is about 45 kDa that contains two parts that 29.991 kDa according to PO and 18 kDa according to pET32a (+)3. The construction of bait plasmids in baterial two-hybrid system and the tests of self-activation.After the construction of bait plasmids of CP and PO, we can screen the cDNA library of sugarcane and thus investigate the function of the protein. The DNA segments of CP and PO were firstly generated by RT-PCR, the target segments were then inserted into pBT plasmids withλcI gene to construct the recombinant bait plasmids pBT-CP and pBT-PO. The bait plasmids were thereafter transformed into competent cells of E. coli XL1-Blue MRF’ Kan-2 and cultured on 30℃to induce the expression of fusion proteins. Since the fusion protein was expressed on very low level and thus could not be detected by SDS-PAGE, we prepared the antiserum ofλcl and utilize it as first antibody to carry out a western blotting. The results suggested that the two fusion protein could express in host cells. We then co-transfromed the bait plasmids and empty pTRG plasmids which would be utilized to construct the prey plasmids into competent cells of E. coli XL1-Blue MRF’Kan-2, and poured the transformants on the plates with or with out 3-AT respectively in order to test the self-activation. The results suggested that both bait plasmids pBT-CP and pBT-PO had no self-activation and thus could be utilized in the further screening of cDNA library.