Micropropagation And Cloning And Quantitative Expression Of Resistant Genes Of The Wild Bananas In Fujian Province

Banana and plantain are the representative plants in gardens in the southern areas of ChangjiangRiver, and the wild banana is becoming more and more attention as the emerging ornamental plant ofplantains. The wild banana resources were widely distributed surrounding Fujian Province, and theywere different in the morphological characteristics and growth habit. The wild banana resources largelydiscovered in Fujian Province are of great importance for studying the origin and evolution of Musaplants, because Fujian province is the northern area of the wild banana distribution. At the same time,the wild banana is tolerance-resistant, especially in cold tolerance, so not only it can be directly appliedto landscaping in cold region, but also it can be used to mine the resistant gene resources such as coldtolerance, and to further modify Musa ornamental plants which are not cold-resistant, extending theapplication of ornamental banana plants to the landscape, and making colder regions can also plantingMusa ornamental plants which are the signature plant of southland scenery. The researches concerningFujian wild banana resources are very few at present, so it is necessary to apply modern biotechnologyto the wild banana’s research. Therefore，this study intends to conduct in vitro culture, gene cloningand qPCR analysis under clod stress in Fujian wild bananas such as the wild bananas from HuanxiTown of Fuzhou City and Sanming City, which will provide a technical support and scientific bases fortheir exploitation and utilization. The main results were as follows:1. The in vitro propagation of Fujian wild bananas.According to category of the Simmonds&Shepherd, the Fuzhou Huanxi wild banana belongedto AB group, but more close to AA group in taxonomic status, which had a certain relationship, but hadobvious differences with other wild bananas in Fujian areas (AA group in‘Fuzhou wild banana’andAB group in ‘Sanming wild banana’), which increased the genetic diversity of wild banana resourcesin Fujian Province.Firstly, the in vitro propagation of sterile system was established from the explants of suckers inHuanxi wild banana and North Hill wild banana in Fuzhou in buds as the explants, and the inductionrates were up to75%after30days.Secondly, the proliferation conditions were optimized using the plantlets subcultured by our lab.①The influence of different light qualities (red, blue, green, white), the different light intensities(0Lx,500Lx,1000Lx,1500Lx, white) on the proliferation were compared in the wild bananafrom Sanming City, and the results showed that the white light under1000lx light intensity was best for porliferation.②The effects of factors of L39（3）on the MS medium adding differentcombinations of NAA (0mg/L,0.1mg/L,0.2mg/L),6-BA (2mg/L,4mg/L,6mg/L) and Ad (0mg/L,15mg/L,30mg/L) on the proliferation were compared in the wild banana from SanmingCity, and the results showed that the best medium for proliferation was MS medium supplementedwith0.1mg/L NAA,4mg/L6-BA, and30mg/L Ad.③The effects of factors of L39（3）on theMS culture medium adding different combinations of NAA (0mg/L,0.2mg/L,0.4mg/L),6-BA(0mg/L,2mg/L,4mg/L) and Ad (15mg/L,30mg/L,45mg/L) on the proliferation werecompared in the wild banana from Sanming City, and the results showed that the best medium forproliferation was MS medium supplemented with0.2mg/L NAA and30mg/L Ad.④Theeffects of factors of L9（33）on the MS culture medium adding different combinations of NAA (0mg/L,0.1mg/L,0.2mg/L),6-BA (2mg/L,4mg/L,6mg/L) and Ad (0mg/L,15mg/L,30mg/L)on the proliferation were compared in the wild banana from Sanming City, and the results showedthat the order of the effect of plant growth regulators on the proliferation of the protocorm-likebody (forming the multiple-bud body) is: Ad＞NAA＞6-BA，and the MS medium adding6-BA6mg/L and Ad30mg/L was the best for the proliferation of the protocorm-like body (forming themultiple-bud body); the order of the effect of plant growth regulators on the proliferation of theprotocorm-like body (forming the big bud) is: NAA＞6-BA＞Ad，and the MS medium addingNAA0.2mg/L and6-BA4mg/L was the best for the proliferation of the protocorm-like body(forming the big bud). The optimized conditions of proliferation for the bud of the Sanming wildbanana were suitable for those of the Fuzhou Huanxi wild banana and Fuzhou Northern Hill wildbanana.Finally, the rooting experiment of the above3wild bananas was conducted, and the resultsshowed that NAA0.4mg/L was best for rooting of the wild bananas, and the survival ratesreached94%after transferring onto the mixture of peat soil, vegetable garden soil and sands(1:1:0.5).2. Cloning of the tolerance-resistant genes from Fujian wild bananas such ascold resistant genes.The total RNA was extracted from the leaves of Fuzhou Huanxi wild banana (M. spp., ABgroup), then the methods of RT-PCR combined with RACE and database of wild banana inMalaysia (M. acuminata, AA group) whole genome sequencing were used to clone the genes, and20cDNA or DNA sequences of tolerance-resistant genes were obtained, which included the maincold resistant genes related to CDPK cold-resistant pathway such as CBF (CBF7-1), ICE1(ICE1-1,ICE1-2, ICE1-3, ICE1-4, ICE1-5, ICE1-6) and HOS1, with the total of8full-length cDNAsequences,6members of KIN10（KIN10-1, KIN10-2, KIN10-3, KIN10-4, KIN10-5, KIN10-6）, which was "the master molecular switch" for stress resistance, full length cDNA and DNAsequences of the resistant genes CHUP1, PSAG1, PSAG2. The accession numbers in GenBank areCBF7-1: KC157575；ICE1-1~6: KC157569, KC157570, KC157571, KC157572, KC157573,KC157574；HOS1: JX678611；KIN10-1~6: KC127685, KC127686, KC127687, KC127688,KC127689, KC127690；PSAG1cDNA: JX317082, PSAG2cDNA: JX317083；PSAG1DNA:JX678610, PSAG2DNA: JX678609；CHUP1cDNA: JX123753, CHUP1DNA: JX880084,respectively.The coding region of the genomic sequence of CHUP1from the Fuzhou Huanxi wild bananacontained8introns and9exons; however,11introns and12exons existed in CHUP1from theMalaysia wild banana, which were remarkably different between them. The genomic sequences ofPSAG1and PSAG2from Fuzhou Huanxi wild banana did not contain introns.3. Analysis of bioinformatics of resistant genes in Fujian wild bananas.Bioinformatics analysis showed that:①the CBF7-1was a hydrophilic stable protein, whichcontained a signal peptide, was located in nucleus according to the sub-cellular localization. It alsohad8phosphorylation sites and the typical conservative domain of AP2/ERF.②the ICE1s werea hydrophilic unstable protein, which contained no signal peptide, and had20-30phosphorylationsites and the specific conservative Helix-loop-helix domain. ICE1s were located in nucleusaccording to the subcellular localization.③The HOS1was a hydrophilic unstable protein, whichdid not contain signal peptide, were located in nucleus according to the subcellular localization. Ithad57phosphorylation sites and the conservative ELYS-like domain.④The KIN10s were ofhydrophilic stable proteins, and KIN10s was located in nucleus or membrane according to thesubcellular localization. KIN10s had21-26phosphorylation sites and13conservative domains,which were very rare, and showed the diversity of KIN10unique function. At the same time, wealso found many conservative domains of KIN10s were of protein-kinase domains, which werethe structure basis for playing the role of “the master molecular switch" in plant stress regulation.⑤The CHUP1was of the unstable hydrophilic protein, which contained a signal peptide, andhad two-dimension transmembrane helices. It had62phosphorylation sites and3conservativedomains: Actin-binding FH2conservative domain, SF0sub-family domain and SignalP-NN（euk）signal peptide. Actin-binding FH2conservative domain was involved in chloroplast of mobilesignal transduction process.⑥The PSAG1and PSAG2were sable hydrophilic proteins, whichdid not contain signal peptide, and they were transmembrane proteins, and located in microbodyor chloroplast. Each of them had4phosphorylation sites and a conservative domain of PSAKconservative family. The homology of PSAG1and PSAG2’s nucleotide sequences and amino acidsequences was high, and the tertiary structure of proteins were similar, which further ensured the stability of PSAG functions by the functional redundancy.4. The expression analysis of qPCR of cold resistant genes in Fujian wildbananas.Low temperature can induce the expression of many genes, which has made the plant toproduce cold tolerance. In this experiment, the in vitro plantlets of Fuzhou Huanxi wild bananawere used as the materials, the relative expression levels of CBF7-1, ICE1s (ICE1-1~ICE1-4),HOS1and KIN10s were detected were treated by qPCR of SYBR Green for verifying thefunctions of these cold resistant genes under the clod stress at the temperatures of0℃,4℃,13℃,20℃,28℃, respectively. The results showed that: The relative expressions of KIN10s and ICE1srose significantly, and achieved the highest at13℃, which indicated that KIN10s and ICE1s of thewild banana expressed largely to enhance the cold resistance of the plant at13℃, which was thecritical temperature and the ordinary banana stopped the growth. At the same time, we also foundthat HOS1’s relative expression level at13℃was slightly higher than that at20℃, but decreaseddramatically when the temperature continued to drop. HOS1’s relative expression level dropped tothe lowest at0℃, but ICE1s’ relative expression level increased obviously at the same time.HOS1has a negative regulatory role to ICE1s, and it would improve the level of ICE1s whenHOS1relative expression level declined. So the cold tolerance of plants would increase. Thesewere the differences of the expression patterns among them, which also accorded with expressioncharacteristics of related genes in CBF cold resistant pathway. In this study, the expression patternof CBF7-1was different from those of KIN10s, ICE1s and HOS1, it declined obviously as thetemperature from28℃to4℃，however，it rose again at0℃in Huanxi wild banana. In a word,the possible molecular mechanism of expressions of cold resistant genes in CBF cold resistantpathway in Fuzhou Huanxi wild banana was as follows：KIN10s expression levels increased Whenunder the low-temperature stress, especially at13℃of the critical growth temperature, whichstarted the "gene networks" of the cold stress response, and the expression level of ICE1sincreased firstly through CBF signalling transduction pathway, and it is inferred that the increaseof ICE1s further results in expressions of COR genes furtherly by signaling transduction of CBF,and finally the cold resistance improves. Furthermore, the expression level of HOS1decreaseddramatically at the low temperature, which could contribute to improving the level of ICE1s, andtherefore the expression levels of COR genes would further rise.