水稻灌浆期耐热相关基因OsHP29和OsHP40的克隆及其功能分析
发布时间：2019-02-16 19:46
【摘要】：全球气候变暖、频繁的夏季高温已成为制约水稻产量和品质的主要气候因子。然而,水稻耐热性受多基因调控、分子调控机制相对复杂。前期研究中,本课题组通过分析水稻应答灌浆期夜间高温的转录组差异,发现了35个可能参与调控水稻灌浆期耐热性的关键转录本,但这些转录本的基因功能尚需进一步的分子验证。为此,本研究采用RACE技术从水稻耐热纯系XN0437T中克隆其中2个关键转录本(OsHP29、OsHP40)的全长序列并进行了生物信息学分析,进一步以pCAMBIA1301为基础载体构建过表达载体并转化烟草,以验证基因OsHP29和OsHP40的分子功能。获得结果如下:(1)基因OsHP29和基因OsHP40的全长分别为235 bp和904 bp;GenBank的BLASTX同源性分析结果显示,它们在数据库中均具有同源序列,但基因OsHP29的功能未知,而基因OsHP40与LOC4329967基因是同源基因,同源基因具有温度诱导脂质运载蛋白的功能。GenBank数据库的ORF finder和软件Protparam分析结果显示基因OsHP29存在一个102 bp的ORF,编码33个氨基酸,其编码蛋白的分子量为3.9334 KDa、PI为12.00,蛋白不稳定系数为109.00;基因OsHP40存在一个588 bp的ORF,编码195个氨基酸,蛋白质分子量为22.2211 KDa,PI为5.22,蛋白不稳定系数为33.90。(2)软件ExPASy、SignalP 3.0 Server、TMHMM Server V2.0和Prot Fun 2.2 Server分析结果显示,基因OsHP29和基因OsHP40各编码一个亲水性蛋白,基因OsHP29编码的蛋白属于不含信号肽的非跨膜蛋白,该蛋白参与调控水稻生长发育、能量代谢过程;基因OsHP40编码的蛋白属于不含信号肽的非跨膜蛋白,是一个结构蛋白,参与蛋白质的结合和转运等过程。(3)过表达载体pCAMBIA1301-OsHP29和pCAMBIA1301-OsHP40通过农杆菌介导的叶盘法转化烟草,基因OsHP29获得了T0代植株12株,经分子检测得到阳性植株6株,阳性率为50%;基因OsHP40获得了T0代植株12株,经分子检测得到阳性植株7株,阳性率为58%。(4)阳性转化植株和野生型植株的耐热性试验数据显示:高温处理2小时结束时,野生型植株叶片发生弯曲,叶片的叶长直线距离由高温处理前的平均5.1 cm弯曲至高温处理结束时的平均3.3 cm,差异达到极显著水平;转OsHP29基因的植株叶片也发生了一定的弯曲,叶片的叶长直线距离由高温处理前的平均5.2 cm弯曲至高温处理结束时的平均3.5 cm,差异达到显著水平;而转OsHP40基因的植株叶片受高温影响较小,叶片的叶长直线在高温处理前、结束时无显著差异,表明目的基因OsHP29和OsHP40具有提高烟草耐热性的功能。
[Abstract]:Global warming and frequent summer high temperature have become the main climatic factors restricting rice yield and quality. However, the heat tolerance of rice is regulated by many genes, and the molecular regulation mechanism is relatively complex. In the previous study, we found 35 key transcripts that may be involved in regulating the heat resistance of rice during grain filling by analyzing the differences of transcriptome during night high temperature in rice response to grain filling. However, the gene function of these transcripts needs further molecular verification. In this study, the full-length sequences of two key transcripts (OsHP29,OsHP40) were cloned from the heat-resistant pure line XN0437T of rice by RACE technique and analyzed by bioinformatics. Furthermore, the over-expression vector was constructed based on pCAMBIA1301 and transformed into tobacco. To verify the molecular function of gene OsHP29 and OsHP40. The results are as follows: (1) the full length of gene OsHP29 and gene OsHP40 are 235 bp and 904 bp;, respectively. The results of BLASTX homology analysis of GenBank showed that they all had homologous sequences in database, but the function of gene OsHP29 was unknown, while gene OsHP40 and LOC4329967 gene were homologous genes. Homologous gene has the function of temperature-induced lipid carrier protein. The results of ORF finder and software Protparam analysis of GenBank database show that there is a 102-bp ORF, encoding 33 amino acids, and the molecular weight of the encoded protein is 3.9334 KDa,. The PI was 12.00 and the coefficient of protein instability was 109.00; The gene OsHP40 has a 588 bp ORF, encoding 195 amino acids, with a molecular weight of 22.2211 KDa,PI and a protein instability coefficient of 33.90. (2) ExPASy,SignalP 3.0 Server,. The results of TMHMM Server V2.0 and Prot Fun 2.2 Server analysis showed that the gene OsHP29 and gene OsHP40 each encode a hydrophilic protein, and the protein encoded by gene OsHP29 is a non-transmembrane protein with no signal peptide, which is involved in the regulation of rice growth and development. Energy metabolism; The protein encoded by gene OsHP40 is a non-transmembrane protein without signal peptide and is a structural protein involved in protein binding and transport. (3) overexpression vectors pCAMBIA1301-OsHP29 and pCAMBIA1301-OsHP40 transform tobacco by Agrobacterium tumefaciens mediated leaf disc method. 12 T0 generation plants were obtained by OsHP29 and 6 positive plants were obtained by molecular detection. The positive rate was 50%. Twelve T0 generation plants were obtained by OsHP40, 7 positive plants were obtained by molecular detection, the positive rate was 58. (4) the heat tolerance test data of positive transformed plants and wild-type plants showed that: at the end of 2 hours of high temperature treatment, The leaf length of wild-type plants was curved, and the difference of leaf length line distance from average 5.1 cm bending before high temperature treatment to 3.3 cm, at the end of high temperature treatment reached a very significant level. The leaf length of the transgenic plant with OsHP29 gene also had a certain curvature, and the leaf length line distance was significantly different from the average of 5.2 cm before the high temperature treatment to the average 3.5 cm, at the end of the high temperature treatment. However, the leaf length of OsHP40 transgenic plants was not affected by high temperature, and the leaf length was not significantly different at the end of high temperature treatment, indicating that OsHP29 and OsHP40 could improve the heat resistance of tobacco.
【学位授予单位】：江西农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S511
本文编号：2424777
[Abstract]:Global warming and frequent summer high temperature have become the main climatic factors restricting rice yield and quality. However, the heat tolerance of rice is regulated by many genes, and the molecular regulation mechanism is relatively complex. In the previous study, we found 35 key transcripts that may be involved in regulating the heat resistance of rice during grain filling by analyzing the differences of transcriptome during night high temperature in rice response to grain filling. However, the gene function of these transcripts needs further molecular verification. In this study, the full-length sequences of two key transcripts (OsHP29,OsHP40) were cloned from the heat-resistant pure line XN0437T of rice by RACE technique and analyzed by bioinformatics. Furthermore, the over-expression vector was constructed based on pCAMBIA1301 and transformed into tobacco. To verify the molecular function of gene OsHP29 and OsHP40. The results are as follows: (1) the full length of gene OsHP29 and gene OsHP40 are 235 bp and 904 bp;, respectively. The results of BLASTX homology analysis of GenBank showed that they all had homologous sequences in database, but the function of gene OsHP29 was unknown, while gene OsHP40 and LOC4329967 gene were homologous genes. Homologous gene has the function of temperature-induced lipid carrier protein. The results of ORF finder and software Protparam analysis of GenBank database show that there is a 102-bp ORF, encoding 33 amino acids, and the molecular weight of the encoded protein is 3.9334 KDa,. The PI was 12.00 and the coefficient of protein instability was 109.00; The gene OsHP40 has a 588 bp ORF, encoding 195 amino acids, with a molecular weight of 22.2211 KDa,PI and a protein instability coefficient of 33.90. (2) ExPASy,SignalP 3.0 Server,. The results of TMHMM Server V2.0 and Prot Fun 2.2 Server analysis showed that the gene OsHP29 and gene OsHP40 each encode a hydrophilic protein, and the protein encoded by gene OsHP29 is a non-transmembrane protein with no signal peptide, which is involved in the regulation of rice growth and development. Energy metabolism; The protein encoded by gene OsHP40 is a non-transmembrane protein without signal peptide and is a structural protein involved in protein binding and transport. (3) overexpression vectors pCAMBIA1301-OsHP29 and pCAMBIA1301-OsHP40 transform tobacco by Agrobacterium tumefaciens mediated leaf disc method. 12 T0 generation plants were obtained by OsHP29 and 6 positive plants were obtained by molecular detection. The positive rate was 50%. Twelve T0 generation plants were obtained by OsHP40, 7 positive plants were obtained by molecular detection, the positive rate was 58. (4) the heat tolerance test data of positive transformed plants and wild-type plants showed that: at the end of 2 hours of high temperature treatment, The leaf length of wild-type plants was curved, and the difference of leaf length line distance from average 5.1 cm bending before high temperature treatment to 3.3 cm, at the end of high temperature treatment reached a very significant level. The leaf length of the transgenic plant with OsHP29 gene also had a certain curvature, and the leaf length line distance was significantly different from the average of 5.2 cm before the high temperature treatment to the average 3.5 cm, at the end of the high temperature treatment. However, the leaf length of OsHP40 transgenic plants was not affected by high temperature, and the leaf length was not significantly different at the end of high temperature treatment, indicating that OsHP29 and OsHP40 could improve the heat resistance of tobacco.
【学位授予单位】：江西农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S511
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