Fine Mapping Of Cold Tolerance Gene QCTB7 And Molecular Mechanism Of CTB4a At Booting Stage In Rice(Oryza Sativa L.)

Low temperature stress is one of important problem of rice cultivation.Low temperature at the booting stage is a significant factor affecting rice production by inducing pollen sterile and reducing spikelet fertility.To exploit the genetic mechanism of cold tolerance at the booting stage,using cold-tolerant NILs developed by backcrossing KMXBG,reported to be the most cold-tolerant rice variety at the booting stage,as donor,with a cold-sensitive Japanese commercial japonica variety,Towada,marker selection and QTL analysis was conducted by NILs analysis combined with BSA and fine mapping of cold tolerance gene was performed.Finally,cold tolerance gene was cloned and the function of protein expressed was studied.This study was divided into two parts:map-based cloning for cold tolerance gene qCTB7 and study on the Functions of cold tolerance protein CTB4a at the booting stage in rice.The main results were as follows:1.BC7F4 population was constructed from NIL 1929 x Towada,qCTB7 was located in a 44.5 kb interval between SSR37-IN7-12 of chromosome 7.Eight putative candidate genes were identified,including six functional genes and two transposons.The sequences of four candidate genes were different between NIL 1929 and Towada.Expression analysis of four candidate genes was performed under different cold treatment period at the booting stage,whinh identified that the levels of two candidate genes expression related to cold treatment.CDS full-length sequence was obtained and sequence analysis was performed.2.RACE amplification of CTB4a was performed,which identified two different transcripts.We predict that CTB4a protein is a leucine-rich receptor-like kinase and hydrophobic transmembrane protein,which consists of extracellular leucine-rich repeat domain and intracellular kinase domain.A KMXBG cDNA library was constructed and the quality was tested,and then,15 CTB4a interacting proteins were screened by yeast two-hybrid using the cDNA library.Through verifying these interacting proteins by bimolecular fluorescence complemen,tation(BiFC),three strong interacting proteins were obtained.According to the forecast results,one is ubiquitin-conjugating enzyme E2(K2)and two were ATP synthase subunit(K4,K5).ATP synthase subunit can interact with CTB4a in the tobacco and rice by CoIP.E2 and ATP synthase subunit as candidate kinase substrates were selected.The results of testing showed that the substrates E2 and ATP synthase subunit can be phosphorylated by protein kinase,which also had the activity of autophosphorylation.ATP synthase activity were also determined in NIL1913 and Towada,while the ATP synthase activity of NIL1913 membrane protein was higher than that of Towada.