The Cloning And Functional Analysis Of The Genes OsHP29 And OsHP40 Related To Heat-tolerance Of Rice At Milky Stage

Global warming and summer short-period high temperature stress has become the main climate factors on restricting rice yield and quality. However, the traits or rice heat tolerance controlled by multiple genes and the molecular regulation mechanism of rice responding to hight temperature is relatively complex. In our previous studies, we analyzed the difference of transcriptomes in rice responding to high night temperature stress at early milky stage. The results showed that 35 transcripts may be involved in rice responding to high night temperature stress. But the function of these genes needs to be further verified.In this study, the full-length sequence of two key transcripts(OsHP29, OsHP40) from rice XN0437 T, were cloned by RACE technology and analyzed by biological information method. The overexpressing vectors based on pCAMBIA1301 were further constructed and transform to tobacco, to verify the molecular function of the gene OsHP29 and gene OsHP40. Results obtained were as follow:(1)The length of OsHP29 gene and OsHP40 gene were 235 bp and 904 bp,respectively. GenBank blastx homology analysis showed that they have homologous sequences in the database, but the function of gene OsHP29 was unknown, OsHP40 and LOC4329967 were homologous genes, the homologous gene has the function of temperature induced lipid transport protein. ORF finder of GenBank database and software Protparam analysis showed that a 102 bp ORF that encoding 33 amino acids existed in the OsHP29 which encoded a protein of molecular weight for 3.9334 KDa, PI 12.00, the protein instability coefficient was 109.00; a 588 bp ORF that encoding 195 amino acids existed in the OsHP40, which encoded a protein of molecular weight for 22.2211 KDa, PI5.22.The protein instability coefficient was 33.90.(2) Software of the ExPASy, SignalP 3 TMHMM, Server V2.0 Server and ProtFun2.2 Server analysis results showed, each of OsHP29 and OsHP40 encoded a hydrophilic protein, the protein encoded by OsHP29 was a non-transmembrane protein that did not contain a signal peptide; this protein involved in the regulation of rice growth and development, energy metabolism process. While the protein encoded by OsHP40 was a non-transmembrane protein that did not contain a signal peptide, which was a structural protein that participates in the binding and transport of proteins.(3)The overexpression vector pCAMBIA1301-OsHP29 and pCAMBIA1301-OsHP40 were transformed into tobacco by Agrobacterium mediated transformation, 12 plants were obtained from OsHp29 gene from T0 generation, 6 positive plants were obtained by molecular detection, its positive rate was 50%; 12 plants were obtained from OsHp40 gene from T0 generation, 7 positive plants were obtained by molecular detection, its positive rate was 58%.(4)The Heat resistant test data of positive transformed plants and wild type plants showed that: At the end of 2 hours of high-temperature treatment, the wild type plant’s leaves became bent, the straight line distance of the leaves were reduced from average 5.1cm to average 3.3 cm, reached an extremely significant level; OsHP29 transgenic plant leaves also had a certain bending, the straight line distance of the leaves were reduced from average 5.2 cm to average 3.5 cm, reached a significant level; OsHP40 transgenic plant leaves were less affected by high temperature, there was no significant difference before and at the end of high temperature treatment. In conclusion, the gene OsHP29 and OsHP40 could improve the heat resistance of tobacco.