Preliminary Functional Analysis Of Sucrose Synthase Gene Family Members (PtSS) In Populus Tomentosa

Wood quality improvement is one of the important research topics in the field of forest tree genetics and breeding, cellulose is closely related to the quality of wood, it is the main components of the cell wall. Cellulose molecule is the glucan chain, which is linked by UDP-G through the beta-1,4 glycosidic links, sucrose synthase (SS) catalyst sucrose into UDP-G and fructose, Wherea UDP-G is the substrates in cellulose synthetic reaction, it plays vital role for glucan chain extension and fiber formation. Therefore, to identify the functions of PtSS family members, to explore the molecular mechanisms of wood formation, are of important theoretical significance, and the potential application value for improvement of wood quality via genetic engineering.In this study, as experimental materials, based on cloning of some PtSS members, and their promoters, as well as completed construction of expression vector of some PtSS members, the nine amiRNAi structures were constructed, furthermore, use Populus tomentosa (TC1521) as receptor material, genetic transformation mediated by Agrobacterium tumefaciens was performed, for a positive plants were screened via PCR identification. Afterward, RT-qPCR, enzyme activity determination and sugar metabolic changes analysis were done respectively. The main results were as follows:1. amiRNAi structures were constructed by using artificial microRNA interference technology, including 6 single-target vectors of amiRNA-PtSS1, amiRNA-PtSS2, amiRNA-PtSS3, amiRNA-PtSS4, amiRNA-PtSS6 and amiRNA-PtSS7, and 3 double-targets vectors of amiRN A-PtSS1-2, amiRNA-PtSS4-5, and amiRN A-PtSS6-7. Furthermore, genetic transformation of amiRNAi structures above metioned was performed in poplar.15 amiRNAi transgenic strains were obtained by PCR identification,90 trangenic plants were propagated.2. The results of RT-qPCR analysis showed that the mRNA (mRNA-PtSSl/PtSS3/PtSS4) expression level of the transgenic lines with corresponding amiRNAi structure (amiRNA-PtSS1/PtSS3/ PtSS4) was downregulated siginificantly in different tissues, other two members in the stems and leaves basicly did not change, while in the root, mRNA of PtSS3 and PtSS4 in amiRNA-PtSS1 transgenic lines increased, mRNA of PtSS4 in amiRNA-PtSS1 transgenic lines increased, mRMA-PtSS1 and PtSS3 in amiRNA-PtSS4 transgenic lines increased too, it may be due to the compensatory effect between different members; As for transgenic plants with overexpression of PtSS (PtSS1/PtSS2/PtSS3), mRNA (mRNA-PtSS1/PtSS2/PtSS3) expression in roots, stems and leaves were upregulated. Those of the other two members did not change.3. The activity of sucrose synthase analysis showed that, compared with wild type, the sucrose synthase activity of three amiRNAi structures significantly decreased in the stems and leaves, but no significant change in the root; The sucrose synthase activity of different transgenic lines of PtSSl, PtSS2 significantly increased in root, stem and leaf tissues, As for transgenic lines of PtSS3, the activities of sucrose synthase obviously increased in stems and leaves, whereas in roots remain the same. In general, the enzymatic activity change characteristics are in conformity with the RT-qPCR analysis results.4. The sugar metabolic analysis indicated that the trend of sugar change of amiRNAi transgenic lines showed the characteristics of sucrose content increase, and fructose and glucose levels decrease in stems and leaves. In roots, sucrose and fructose content basically remain unchanged in amiRNA-PtSS1 and amiRNA-PtSS3 lines, in the root of transgenic lines of amiRNA-PtSS4, sucrose content decreased, fructose content increased; but the glucose contents of transgenic plants of all three amiRNAi structures are reduced. In the leaves, the amount of glucose of amiRNA-PtSS3 and amiRNA-PtSS4 lines unchanged; As for transgenic lines with PtSS members, sugar content generally presents the characteristics of sucrose content reduce, fructose and glucose increase. In the roots of PtSS3 transgenic lines, there was no obvious changes of sucrose, fructose and glucose. Because PtSS3 is single copy in poplar genome, there may be a feedback inhibition in root tissue. In the leaves of PtSS2 transgenic lines, glucose content remains the same.