Cloning And Preliminary Functional Verification Of Gene With Cold Tolerance For Xinjiang Bermudagrass

Xinjiang bermudagrass can endure the low temperature of-32? when under snow, which is an excellent germplasm resource in warm-season turfgrasses. This study obtained cold-related differential fragments of bermudagrass by BSA based on associated sites that come from association analysis and cloned and preliminary function verification gene with cold tolerance of bermudagrass combined in silico cloning and RT-PCR to explore its mechanism of cold-resist and tap its gene with cold tolerance. The main results are as follows.The known cold-related sits that come from association analysis were amplified in the cold tolerance and cold sensitive gene pool of bermudgrass, then obtaining and analyzing the cold-related fragments. The results indicatied that 43 differential fragments were obtained. Moreover, 26 differential fragments were sequenced successfully. Among that, 17 sequences could find homologous sequence, of which 14 relate with metabolic enzymes and proteins and 3 sequences were unknown sites. 12 sequences could not find homologous, which may be some new genes. There were high homolog between the sequence ES8174-2with rice LTI6 A, Zea mays hydrophobic protein LTI6 A and Pennisetum hypothermia and salt responsive protein, with the identity being 94%?90% and 93%.Bermudagrass LTI6 A gene was cloned by the methods of in silico cloning and RT-PCR in Xinnong-1bermudagrass based on the sequence ES8174-2, which had a consistent sequence of 93% with electronics sequence. Forecasting the function of the obtained gene by bioinformatics analysis and the results indicated that the protein molecular weight was 6229.6 Da and theoretical pI: 4.56, atomic composition:C306H462N60O70S4, measured 14105, the instability index: 40.11, grand average of hydropathicity: 1.509,aliphatic index: 141.07. It had two transmembrane and no signal peptide. It was hydrophobin and located in cell membrane. Function prediction demonstrated that the protein coded by bermudagrass LTI6 A gene played the same function as the protein coded by OsLTI6 A gene did.The bermudagrass LTI6 A was recombined in the expression vector pCAMBIA1301 to construct an expression vector for bermudagrass LTI6 A gene. Then it was transited into the tobacco genome by agrobacterium-mediated method. When the transgenic tobacco plants were under low-temperature stress treatment, its relative electric conductivity was lower than the control transgenic tobacco's. Afetr being returned to normal temperature, the transgenic tobacco plants did not develop frozen spots. We can indicate that the gene can reduce electrolyte leakage and improve the stability of the membrane. The result preliminary indicates that bermudagrass LTI6 A gene can increase the cold resistance of plants.