Analysis Of Differentially Expressed Genes Associated To Starch Biosynthesis Between Xushu18 And Xu781 And Cloning Of IbFBA Gene

RNA-seq is an easy and rapid method to obtain functional genes, and to estimate the expression level of the genes according to the sequencing. In this study, we got 14 differentially expressed genes which were related to starch biosynthesis from RNA-seq data of Cv. Xushu18 and Xu781. In order to understand the functions of these genes, to analyze the correlation between gene expression and starch content, quantitative RT-PCR method was used to test the expression pattern of them in the expansion period of sweetpotato. Then we cloned 2 genes which were named IbFBA2 and IbFBA5 and preliminary verified the functions of them.1. We found there were 6 gene expression patterns of the 14 genes by qRT-PCR method, there were increasing persistently of gene expression, reducing gradually of gene expression and so on. The expression patterns of same gene were different between 2 varieties, as well as same gene expression in different tissues of one variety. For example, NMT3 showed 4 expression patterns in leaf and root of Cv.Xushu18 and Xu781. Generally, other genes showed 2 or 3 different expression patterns.2. We tested the starch and soluble sugar contents of the samples which sampled for qRT-PCR analysis from 4 times of expansion period of Cv. Xushu18 and Xu781, simultaneously. The change of starch content was same to the expression pattern of IbFBA2 in Xushu18 leaf, also same to expression pattern of IbFBA2, NMT3, SBPase, XYL1, T1-23698 and T2-33205 in Xu781 leaf. The expression levels of these genes and starch contents were reduced firstly and then increased. The change of starch content was same to expression pattern of GLDP1, IbAGPb1 A, SBPase, T1-30106 and T2-33205 expression pattern in Xushu18 root. The expression levels of these genes and starch contents were gradually increased. But the change of starch content was same to expression pattern of IbAGPb1 A, IbFBA5, MAP65-1, NMT2, T1-23698 and T2-33205 in Xu781 root. The expression levels of these genes and starch contents were raised firstly then reduced. The results showed that the expression pattern of IbFBA2 was in harmony to starch content in leaves of Xushu18 and Xu781, and the expression patterns of IbAGPb1 A and T2-33205 were in harmony to starch content in root of these 2 cultivars. Moreover, gene expression pattern of XYL1 was same as sucrose contents of whole growth period, both of them were from high level to low level.3. IbFBA2 and IbFBA5 were cloned from Cv. Xu781. The full length of the IbFBA2 cDNA was 1617 bp with an open reading frame of 1197 bp, which encodes 398 amino acid residues, containing 5 extrons and 4 introns. It was located in plasmid for subcellular localization by bioinformatics prediction. The full length of the IbFBA5 cDNA was 1341 bp with an open reading frame of 1074 bp, which encodes 357 amino acid residues, containing 3 extrons and 2 introns. It was located in cytoplasm for subcellular localization by bioinformatics prediction.4. We analyzed the expression level of IbFBA2 and IbFBA5 in 7 tissues of Xu781 which sampled 70 days after planting. The results showed that IbFBA had different expression level in 7 tissues. IbFBA2 was notablely high expressed in upper of sweetpotato, low expressed in root. IbFBA5 was highly expressed in leaf, storage root, pencil root, and low expressed in flower.5. Both of IbFBA2 and IbFBA5 were response to salt, simulated drought and ABA treatment, however, with different expression patterns. The expression level of IbFBA2 was usually decreased to adapt the stress, and then increased at the later stage. For IbFBA5, the expression level was directly increased.6. We injected tobacco leaves with Agrobacterium(EHA105) which carried overexpression vector of pGWB12::IbFBA2 and pGWB12::IbFBA5 and dyed with iodine. After 3 days we detected the injected leaves to check whether there was starch formation. However, we didn’t get the expected results, and inferred that IbFBA genes might be not the major genes of starch biosynthesis. According to complementary experiment of transgenic Arabidopsis, we found that Arabidopsis mutant which transformed IbFBA5 was response to ABA treatment, and the root lengths of transgenic plants were restored to wild-type phenotype.