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RAPD Analysis, in Vitro Culture and Cloning of SOD Gene of the Banana (Musa Spp.) Germplasm in Fujian Province

Author: ZhangRui
Tutor: LaiZhongXiong
School: Fujian Agriculture and Forestry University
Course: Pomology
Keywords: wild banana RAPD in vitro conservation gene cloning
CLC: S668.1
Type: Master's thesis
Year: 2011
Downloads: 59
Quote: 6
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Abstract

China is not only one of the originating centers of Musa but also has an abundance and diverse germplasm resources.In the long process of evolution, gradually formed a natural gene pool, which contains the wild species of Musa.To provide a material basis for genetic improvement of bananas. Fujian is rich in resources of wild banana. In view of this, genetic polymorphism of wild banana germplasm resources, which were collected around Fujian Province, was analyzed by the RAPD molecular marker. Then the in vitro conservation of partical wild banana germplasm resources was conducted. Mn-SOD gene from the leaf of wild banana plantlets was cloned, which laid the foundation for the study of the classification and identification, origin and evolution and in vitro conservation of wild banana.The main results were as follows:1. RAPD analysis on wild banana resources from some areas of Fujian Province7 accessions samples of wild banana resources(Sanming wild bananas, Minhou Sandiejing wild bananas,Youxi wild bananas, Fuzhou Qishan wild bananas, Taiwan introduction wild bananas, Wuyi Mountain wild bananas) from some parts of Fujian Province were used as materials for RAPD analysis.The results showed that: 183 RAPD bands were amplified by 13 random primers, including 153 polymorphic bands. The ratio of polymorphic bands was 83.61%. According to the clustering analysis results: the genetic similarity coefficient of 7 accessions of wild bananas resource was at 0.448 - 0.833. At D1=19.0 district, 7 accessions of wild banana resources were divided into two distinct groups, and at D2 = 16.5 the first group was divided into two subgroups, at D3 = 4.95 the first sub-group was divided into two small groups; the second group was divided into three subgroups.2. Analyses of the genetic diversity of 3 populationsThe genetic diversity and cluster analysis from 85 individuals of 3 clusters (population 1——Minhou18-streams wild banana; population 2——Yuxi wild banana; population 3——Sandiejing wild banana) were analyzed using POPGENE32 software .The results showed that, the effective number of alleles in population 1 was 1.1291±0.2705, Nei’s gene diversity index was 0.0782±0.1533, and Shannon’s information index was 0.1199±0.2250;The effective number of alleles in population 2 was 1.2218±0.3284, Nei’s gene diversity index was 0.1337±0.1811, Shannon’s information index was 0.2051±0.2626);The effective number of alleles in population 3 was 1.2176±0.3239, Nei’s gene diversity index was 0.1318±0.2025, and Shannon’s information index was 0.1795±0.2608. The total population of the effective number of alleles was 1.4499, Nei’s gene diversity index was 0.1904, and Shannon’s information index was 0.2628. These results showed that the three populations had relatively high genetic diversity .and genetic differentiation coefficient was Gst0.5635 among the three populations.It suggested that the genetic variation among the three populations mainly existe in populations.3. In vitro conservation of wild banana germplasm in Fujian ProvinceThe multiple shoot of Fuzhou and Sanming wild banana plantlets coubld be conserved 9 months, Fuzhou banana could be conserved for 6 months and not etiolated death, which were conserved on MS + 6-BA1.0 mg / L +NAA0.1 mg / L + sucrose 3% + Ago.6% medium, at the temperature controlled at 22℃with the light intensity 1600 Lux,12h / d conditions.4. Mn-SOD gene cloning from the plantlet leaf of Fuzhou wild bananaThe young leaves of Fuzhou wild banana in vitro plantlets were used as materials for cloning the Mn-SOD gene. The full-length cDNA sequence of Mn-SOD gene was obtained 831bp.It was registered in GenBank, and accession numbers was JF752346.The Mn-SOD gene contained 137bp5’UTR, 151 bp 3’UTR, and 3’-end involved 17 poly (A) tails. The sequence of this gene was highly homologous with that other plants reported in GenBank.The cDNA contained a 543 bp open reading frame, encoded 181 amino acids, with ATG as start codon and TGA as stop codon.The predict of protein’s physical and chemical properties showed that, the relative molecular weight of protein Mn-SOD was 20108.8Da; the isoelectric point (pI) was 7.92; the molecular formula was C917H1402N248O259S2; the total number of atoms was 2828; the negatively charged residues (Asp+ Glu) was 18 and the positively charged residues (Arg+Lys) was 19; Because hydrophilic amino acids were more than hydrophobic amino acids, the whole polypeptide chain performance was hydrophilic polypeptide. The protein consisted of 20 amino acids, which had more abundant of Ala, Leu, Lys, Gly, Val, Asn, Glu,was 20 (11.1%), 18 (10.0%), 16 (8.9%), 15 (8.3 %), 13 (7.2%), 12 (6.7%), 10 (5.6%), respectively. Met was the lowest with only 2 (1.1%). The protein is a hydrophilic protein, which didn’t contain signal peptide and transmembrane domains. The the maximum credibility that its sub-cellular localization was in the mitochondrial was 0.640.Secondary structure prediction showed that the Mn-SOD protein was with 51.11%α-helices 8.89%β-strands and40.00% irregular curl. Looking at the overall structure of the protein, the main structural elements of wild banana in vitro plantlets Mn-SOD protein wereα-helices and irregular curl, and which were spreaded into the whole protein.Three-dimensional structure prediction showed that wild banana in vitro plantlets Mn-SOD protein was surrounded with 4β-fold and5α-helices . These studies were laid the important foundation for the study of SOD mechanism during wild banana in vitro plantlets conservation.

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