Isolation And Polymorphism Analysis Of Full Length Sequence Of Sucrose Sucrose Synthase(AhSS2) Gene From Jackfruit

Jackfruit is a common tropical fruit,and it’s sugar mainly consists of glucose,fructose and sucrose.One of the key enzymes accounting for sugar accumulation during fruit ripening is sucrose synthase.In this thesis,full sequence of the sucrose synthase(AhSS2)gene was cloned by RACE technique from jackfruit,and bioinformatics analysis were carried out;also polymorphisms of sequences among different jackfruit germplasm were evalued.The main findings were as follows:1.The full sequence of the cDNA of the AhSS2 gene was cloned by RACE,and the length of ORF was 2436 bp,located between 148 nt and 2583 nt,encoding 811 amino acids.The AhSS2 gene encodes a protein with a molecular weight of 92.583 kDa and an isoelectric point pI of 5.77,which is a hydrophilic acidic protein.There are two main domains in the protein,the first is sucrose synthase site located between 8-555 nt,the second is the glycosyltransferase site located between 558-745 nt.Bioinformatics analysis indicated that there is no signal peptide as well as transmembrane domain.The secondary structure and the three-dimensional structure of the protein were also analyzed.2.Using the total genomic DNA,primers were designed according to the exon sequence,and the full length of the AhSS2 gene and the exon 1-2 and intron 1 sequences of AhSS1 were obtained.The AhSS2 gene contains 15 exons,and exon 1-15 is 96 bp,131 bp,156 bp,193 bp,119 bp,219 bp,94 bp,172 bp,115 bp,172 bp,224 bp,322 bp,244 bp,141 bp and 28 bp.Fourteen introns are found in the gene,and the intron 1-14 are 127 bp,453 bp,110 bp,228 bp,146 bp,170 bp,Bp,301 bp,88 bp,172 bp,412 bp,93 bp,82 bp and 123 bp,respectively.3.The polymorphisms of DNA sequence among 45 jackfruit germplasm were analyzed using several exons and intron sequences of AhSS2 and AhSS1.The genetic similarity coefficients of these germplasm based on sequences of exon 1-7 of AhSS2 gene were 99.53 %,99.40 %,98.06 %,99.94 %,95.92 %,98.04 % and 97.20 %,respectively.The genetic similarity coefficients of these germplasm based on sequences of exon 1 and 2 of AhSS1 gene were 93.84 % and 99.62 %,respectively.The similarity coefficients of intron 2-6 of AhSS2 were 84.79 %,91.35 %,96.51 %,88.85 % and 91.34 %,respectively.The genetic similarity coefficient of these germplasm based on sequences of the intron 1 of the AhSS1 gene was 84.98 %.The exons 1 and 2 of AhSS2 gene are involved in the sucrose synthase domain,and the exons 4 and 5 of AhSS2 gene are involved in the glycosyltransferase domin.4.The length of exon 1-7 of AhSS2 gene was 109 bp,104 bp,178-179 bp,73 bp,223-227 bp,243-245 bp and 140-142 bp.The length of exon 1-2 of AhSS1 was 39-41 bp and 52 bp,respectively.The exon 2 of the AhSS2 gene had 10 SNPs;exon 2 had 8 SNPs;exon 3 had 13 SNPs,1 INDEL;exon 4 had 2 SNPs;exon 5 had 25 SNPs,3 INDELs;exon 6 had 17 SNPs,2 INDELs;exon 7 had 12 SNPs,2 INDELs.Exon 1 of AhSS1 gene had 4 SNPs,2 INDELs;exon 2 had 7 SNPs.The length of intron 2-6 of AhSS2 was 427-488 bp,398-411 bp,92-93 bp,81-83 bp and 192-216 bp,respectively.The length of intron 1 of AhSS2 was 192-220 bp.The results showed that there were 145 SNPs and 22 INDELs in the intron 2 of the AhSS2 gene.There were 74 SNPs and 12 INDELs in the intron 3,and 8 SNPs and 1 INDEL in the intron 4.There are 19 SNPs and 2 INDELs in intron 5,28 SNPs and 9 INDELs are found in intron 6.Intron 1 of AhSS1 gene has has 54 SNPs,13 INDELs.