Cloning And Analysis Of Banana Bunchy Top Virus Genomes And DNA2ORF, And Self-activation Verification Of DNA2ORF In Yeast Two Hybrid

Banana is an important tropical and subtropical fruit. It has become more than4hundreds million of120countries and areas of food and sources income. Banana bunchy top disease was crisis of destroys in banana, special in20century, it threaten one forth of the world banana industry including Asia, Africa and the South Pacific. BBTD was caused by banana bunchy top virus, belonged to Nanaviridae family Babuvirus genus in2005(ICTV). Now, we determined that the virus particle consisted18～20nm of mitochondria diameter, genome composition with at least six ssDNA about1.0-1.1kb. Currently, the amount of BBTV genomes sequence in NCBI GenBank is limited and rarely reported research of protein function. This thesis using the tender pseudostem and leaves of banana infected BBTV (Haikou2and Haikou4) from Haikou, Hainan, China, extraction total DNA as a template, amplifing satellite DNA from BBTV Haikou2and Haikou4isolates by PCR and further amplifing genomes of two BBTVs. Using various of bioinformatic softwares prediction of structural characteristics, gene encoding protein function of Haikou2and Haikou4genomes. In order to identify DNA2ORF, DNA2UTR and the size of DNA2protein of BBTV, we using Race technology to analysis. At last, construction Haikou4BBTV DNA2ORF to bait plasmid of yeast two hybrid (ProQuestTM Two-Hybrid System, Invitrogen), activation verification, the results indicate that weak growth on25mM of3-AT concentration of Sc-Leu-Trp-His plate, but pDESTTM32-DNA2ORF/MaV203un-growth on50mM of3-AT concentration of Sc-Leu-Trp-His plate. The bait plasmid can be inhibited on the concentration of50mM3-AT, thus,50mM of3-AT of Sc-Leu-Trp-His plate can be used for screening yeast two-hybrid with bananas total cDNA library. This research is providing basis for the world’s tropical and subtropical regions of phylogenetic relationships of BBTV isolates and laying some preliminary work for BBTV DNA2protein-coding function. The main conclusions of this paper are as follows:1. Cloning BBTV Haikou2and Haikou4genomes by PCR and Reverse-PCR. The result shows that, the full-length sequence of HaiKou2DNA1-6, Satellite DNA were1103,1067,1058,1041,1013,1082and1093nt, respectively, HaiKou4DNA1-6full-length sequence as1103,1055,1059,1041,1013and1081nt, respectively. Each component of non-coding region of two isolates had one high homologous CR-SL and CR-M; coding region had a open reading frame (ORF), and a small ORF was existed inside DNA1large ORF. Sequence analysis shows that DNA1component of two isolates are the most conservative as compared with others. DNA2component has maximum variation. Because South-pacific group BBTV expanded to the area of Africa and BBTV limited in Southeast Asian. We further divided BBTV to new group, Southeast Asian group and Pacific-Indian ocean group. It was found that Haikou2and Haikou4isolates were the member of Southeast Asian group.2. It was found that BBTV Haikou2isolate exited satellite DNA component, encoding33.1kD Replication initiation protein, while Haikou4isolate is not existence. The full-length of satellite DNA UTR is about238bp, consisting12of motifs, including TATA box, archA, archB, etc. regulation sites. But it was difference with CR-CL and CR-M of DNA1-6. Rep regulated its satellite replication, while mRep regulated DNA1-6replication. Using bioinformatics software, analyzing and forecasting of physical and chemical properties, composition, signal peptide, phosphorylation, secondary structure, tertiary structure and functional domains of the protein, etc., comparing with BBTV DNA1Rep. This is the basis important foundation of Hainan BBTV satellite DNA depth study.3. Using Hainan banana samples infected BBTV Haikou2and4isolates, extraction total RNA, transcript into ds cDNA, the experiment made determining of BBTV Haikou2and4DNA2ORF by3’and5’Race. Comparing with Australia BBTV isolate, we found that mutation of5’terminal, the start site of coding moved downstream168bp of comparing with Australia isolate DNA2. After mutation, the size of ORF is96nt, encoding32amino acid of mini-protein, which lost56amino acid in the front of the5terminal (causing most of the amino acid N-terminal absence). However, even though lost of most amino acid in this mini protein, its function was still exercising or playing a role in biology.4. Construction Haikou4BBTV DNA2ORF to bait plasmid of yeast two hybrid (ProQuestTM Two-Hybrid System, Invitrogen), activation verification, the results indicated that weak growth on25mM of3-AT concentration of Sc-Leu-Trp-His plate, pDESTTM32-DNA2ORF/MaV203un-growth on50mM of3-AT concentration of Sc-Leu-Trp-His plate, the bait plasmid could be inhibited on the concentration of50mM3-AT, thus,50mM of3-AT of Sc-Leu-Trp-His plate can be used for screening yeast two-hybrid with bananas total cDNA library. This research was provided basis for the world’s tropical and subtropical regions of phylogenetic relationships of BBTV isolates and layed some preliminary work for BBTV DNA2protein-coding function.