Cloning And Expression Analysis Of Cold Resistance Relative Genes Of The Wild Banana (Musa Spp., AB Group)

In the experiment, the leaves of the wild banana (Musa spp., AB group, from Sanming City) under normal temperature and cold stress were used as materials for cloning the chitinase andβ-1,3-glucanase genes for obtaining antifreeze protein genes. The rules of gene expression during normal temperature and cold stress were analyzed by real time fluorescence quantitative PCR (q-PCR) for comfirming antifreeze protein genes and revealing the expression difference of the two genes during normal temperature and cold stress. The main results were as follows:1 Cloning and bioinformatics analysis of the cDNA of chitinase genes(ChiⅠ1 and ChiⅠ2) of the wild banana from Sanming City under cold stressFull length cDNA (1097bp) of the chitinase gene was cloned from the wild banana leaves under cold stress (4℃64h) by RT-PCR combined with RACE. It contained 149bp in the 3'UTR and 3'-end including 17bp poly (A) tail, named as ChiⅠ2 (GenBank:FJ222750). Meanwhile, a full length of cDNA (1115bp) of chitinase gene was cloned from the wild banana leaves under the normal temperature (28℃), containing 14bp 5'UTR and 149bp 3'UTR and 3'-end including 17bp poly (A) tails, named as ChiⅠ1 (GenBank:FJ858155). The above two genes were highly homologous to corresponding genes of other plants reported in the database of NCBI. The results which were analyzed by using bioinformatic methods showed that the two proteins were acidic hydrophilic proteins with transmembrane domain and mainly located outside. They belonged to the chitinases family 19, chitinase classⅠ. ChiⅠ2 contained two chitin-binding domains, which maybe the main function domains combining antifreeze activity with antivirus activity. The structure of Chi 12 gene of the wild banana from Sanming City was similar to chitinase antifreeze protein which had reported in the world. Accordingly, Chi 12 gene was preliminary inferred to as a chitinase-AFP gene.2 The cloning of ChiⅠ1 and ChiⅠ2 genomics of the wild banana from Sanming CityThe DNA sequences of ChiⅠ1 and ChiⅠ2 genes of the wild banana from Sanming City were cloned, which ATG was the initiation codon and TAG was the stop codon. The full sequences of Chi 12 were 1183bp (GenBank:GU391234) and the full sequences of ChiⅠ1 were 1159bp (GenBank:GU391235). Sequence analyses indicated that ChiⅠ1 and ChiⅠ2 genes both contained four exons and three introns, which splicing sites obeyed with the GT-AG rule. The first exon of Chi 12 has 24bp more than the first exon of ChiⅠ1, which maybe the antifreeze site of chitinase-AFP. There was no difference of other exons and introns between ChiⅠ1 and Chi 12.3 Promoter cloning of ChiⅠ1 and ChiⅠ2 of the wild banana from Sanming CityThe promoter sequences of ChiⅠ1 and ChiⅠ2 of the wild banana from Sanming City were cloned by genomic walking methods of thermal asymmetric inter-laced PCR. The length of ChiⅠ1 promoter was 323bp (GenBank:GU391235) and the length of Chi 12 promoter was 846bp (GenBank:GU391234). The promoter of ChiⅠ1 had a likehood of basic promoter area from 272bp to 322bp with a predicted starting site of transcription as Cytonsine on the 312th base, and it contained some cis-action elements such as a module involved in light responsiveness, cis-acting element involved in salicylic acid responsiveness, and a cis-acting element conferring high transcription levels. The promoter of Chi 12 also had a possible basic promoter area from 522bp to 572bp with a predicted starting site, adeine on the 562th, containing some cis-action elements such as a module involved in light responsiveness, cis-acting regulatory element essential for anaerobic induction, fungal elicitor responsive element, cis-acting regulatory element involved in the MeJA-responsiveness, gibberellin-responsive element, cis-acting element involved in salicylic acid responsiveness, cis-acting element conferring high transcription levels and cis-acting regulatory element involved in circadian control. The fungal elicitor responsive element, which mainly existed in the promoter of pathogenesis-related protein genes and the element of promoter stress response, was very critical for inducing fungal response. The element was involved in MeJA-responsiveness associated with cold hardiness, which indicated that Chi 12 of the wild banana from Sanming City maybe a chitinase antifreeze protein gene relative to cold resistance and antivirus.4 The expression of chitinase gene of the wild banana from Sanming City under low temperature stressUsing 18S rRNA as the reference gene, the expressions of ChiⅠ1 and ChiⅠ2 under cold stress were analyzed by q-PCR. The results showed that Chi 12 had the most abundant expression under treatment of 15℃for 30h. At 4℃, the expression of Chi 12 increased at the beginning of the cold stress treatment, then declined and finally increased at the end of 24h cold stress treatment. ChiⅠ1 had the most abundant expression under 24h 4℃treatment, with the whole procession of ChiⅠ1 expression presented as a 'M' curve. The results confirmed that Chi 12 was sensitive to temperature variation. Generally, the critical temperature of banana growing was 15℃. When the wild banana was treated under 15℃, Chi 12 gene responded to injury via low temperature, as seen in the high expression levels. The expression under 4℃indicated the Chi 12 gene had a cumulative effect. The expression showed that ChiⅠ1 gene was insensitive to temperature and cold stress time, only when the low temperature continued for some time, was the gene expressed. All these showed that Chi 12 gene of the wild banana was a chitinase-AFP gene, and ChiⅠ1was the isozyme of ChiⅠ2.Also, activity of exo-chitinase and endo-chitinase from the wild banana were measured, the results showed the activity of both genes were strongest after 64h 4℃treatment, which was supposed to be induced by the expression of the antifreeze gene from the chitinase family under cold stress.5 cDNA cloning and bioinformatics analysis ofβ-1,3-glucanase genes (gsp and gspl) of the wild banana from Sanming City under cold stressFrom the wild banana leaves under 64h 4℃treatment, the ORF ofβ-1,3-glucanase gene with a cDNA length of 915bp, encoding 304 amino acids, obtained by RT-PCR combined with RACE technology, named as gsp (GenBank:FJ858156). Meanwhile, an ORF of 951bp cDNA ofβ-1,3-glucanase gene was cloned from the wild banana leaves at the normal temperature (28℃), encoding 316 amino acids, was named as gsp1 (GenBank:HQ018802). The sequences of gsp and gspl gene of the wild banana were significantly homologous with that of other banana varieties in GenBank. There was a 36bp deletion in the N-terminal of gsp in contrast with gspl. The results which were analyzed by bioinformatic methods showed that GSP and GSP1 belonged to glycosyl hydrolases family 17, which were basiCityβ-1,3-glucanase. The two proteins mainly located at cytoplasm and plasma membrane, respectively. GSP protein was a kind of hydrophilic protein, but GSP1 protein was the kind of hydrophobic protein. In contrast to gspl, gps had a 36bp deletion in N-terminal. Preliminarily, the gsp gene was thought to be an antifreeze-protein gene.6 DNA cloning of gsp and gspl genes of the wild banana from Sanming CityThe DNA sequences of gsp and gspl genes of the wild banana from Sanming City were cloned. The full sequences of gsp were 1540bp (GenBank:HQ018804), and the full sequences of gspl were 1531bp (GenBank:HQ018803). Sequence analyses indicated that gsp contained a 625bp intron, which splicing sites didn't obey with the GT-AG rule. And gspl contained a 580bp intron, which splicing sites obeyed with the GT-AG rule. The differences of the intron between the two genes were most likely to the differences betweenβ-1,3-glucanase antifreeze protein gene and commonβ-1,3-glucanase gene.7 The expression ofβ-1,3-glucanase gene of the wild banana from Sanming City under cold stressUsing 18S rRNA as the reference gene, the transcriptional expression levels of gsp and gspl genes in the low-temperature treatment process were analyzed by q-PCR. The result showed that the highest expression of gsp gene of the wild banana was at 15℃treated 30h, the trend of changes was similar to Chi 12 gene of the wild banana. Though the expression of gspl gene varied unconspicuously under cold stress. All these showed that gsp and Chi 12 gene of the wild banana could respond to cold stress rapidly and had cumulative effect. The results in further explanation showed that gsp gene maybeβ-1,3-glucanase antifreeze protein gene.In summary, in the study the chitinase andβ-1,3-glucanase genes with characteristics of cold resistance were cloned from the wild banana from Sanming City under cold stress. Based on the molecular weight, structure and the expression under cold stress, Chi 12 and gsp were probably deduced to be antifreeze protein genes. This information would provide target gene for cold-resistant gene-engineering of banana and other tropical and subtropical crops. Simultaneously, the promoter sequences of Chi I land Chi 12 were cloned, which might lead to reveal the mechanism of cold-resistance and the expression and regulation of cold-resistance genes of the wild banana.