Genetic Transformation Of Sugarcane Sucrose Transporter Gene SoSUT5

Sugarcane is the most important sugar and energy crop in the world. Sugar production from sugarcane accounts for 92%-94% of the total sugar production in China. Breeding new sugarcane varieties with high yield, high sugar or strong tolerance to adverse environments is an important foundation to ensure sustainable development of sugar industry. For sugarcane improvement, conventional breeding is still the main way to obtain new varieties. However, genetic engineering is showing obvious advantages, and its application is more and more popular. In this study, a sugarcane sucrose transporter gene SoSUT5 was used as an exogenous gene to construct monocotyledon or dicotyledon expression vectors. And the genetic transformation mediated by Agrobacterium tumefaciens has been done in both sugarcane and tobacco. The main results were as follows.1. Genetic transformation in tobacco. The dicotyledon expression recombinant vector pRI 101-ON-SoSUT5 was constructed, and the T-DNA region on the vector was transformed into leaf discs of tobacco variety K 346 mediated by Agrobacterium tumefaciens. Thirty one plants of regenerated tobacco were obtained through tissue culture. All of them were detected with PCR. The npt II and target genes were amplified with PCR, and the transformation efficiency was 100%(npt Ⅱ+) and 93.55% (SoSUT5+), respectively. The soluble sugar content in the wild type, pRI 101-ON transgenic type and pRI 101-ON-SoSUT5 transgenic type were determined, which showed that in pRI 101-ON-SoSUT5 transgenic type> pRI 101-ON transgenic type> wild type.2. Construction of RNA interference vector. In this study, Target Finder SiRNA prediction software was used to analyze siRNA interference fragments included in SoSUT5 gene, and a 550 bp sequence was chosen as the interference fragment. The fragment was inserted into pTCK 303 interference vector in forward and reverse orders, respectively. At the same time, Hyg concentration selection was done for resistance screening in sugarcane variety B8. It was found that the optimal Hyg concentration for resistance screening was 50 mg/L at both callus induction and differentiation stages while 40 mg/L at rooting stage.3. Genetic Transformation in sugarcane. The monocotyledon expression vector pUBTC-SoSUT5 was constructed, and the T-DNA region on the vector was transferred into the callus cells of sugarcane variety B8. Through tissue culture and resistance screening under PPT 2.5 mg/L,128 plants regenerated in 48 days. The marker gene bar in the transgenic plants was detected with PCR.91 plants were found positive among the total 128 transformed plants detected.