水稻灌漿期耐熱相關(guān)基因OsHP29和OsHP40的克隆及其功能分析
發(fā)布時(shí)間：2019-02-16 19:46
【摘要】：全球氣候變暖、頻繁的夏季高溫已成為制約水稻產(chǎn)量和品質(zhì)的主要?dú)夂蛞蜃印Ｈ欢?水稻耐熱性受多基因調(diào)控、分子調(diào)控機(jī)制相對復(fù)雜。前期研究中,本課題組通過分析水稻應(yīng)答灌漿期夜間高溫的轉(zhuǎn)錄組差異,發(fā)現(xiàn)了35個(gè)可能參與調(diào)控水稻灌漿期耐熱性的關(guān)鍵轉(zhuǎn)錄本,但這些轉(zhuǎn)錄本的基因功能尚需進(jìn)一步的分子驗(yàn)證。為此,本研究采用RACE技術(shù)從水稻耐熱純系XN0437T中克隆其中2個(gè)關(guān)鍵轉(zhuǎn)錄本(OsHP29、OsHP40)的全長序列并進(jìn)行了生物信息學(xué)分析,進(jìn)一步以pCAMBIA1301為基礎(chǔ)載體構(gòu)建過表達(dá)載體并轉(zhuǎn)化煙草,以驗(yàn)證基因OsHP29和OsHP40的分子功能。獲得結(jié)果如下:(1)基因OsHP29和基因OsHP40的全長分別為235 bp和904 bp;GenBank的BLASTX同源性分析結(jié)果顯示,它們在數(shù)據(jù)庫中均具有同源序列,但基因OsHP29的功能未知,而基因OsHP40與LOC4329967基因是同源基因,同源基因具有溫度誘導(dǎo)脂質(zhì)運(yùn)載蛋白的功能。GenBank數(shù)據(jù)庫的ORF finder和軟件Protparam分析結(jié)果顯示基因OsHP29存在一個(gè)102 bp的ORF,編碼33個(gè)氨基酸,其編碼蛋白的分子量為3.9334 KDa、PI為12.00,蛋白不穩(wěn)定系數(shù)為109.00;基因OsHP40存在一個(gè)588 bp的ORF,編碼195個(gè)氨基酸,蛋白質(zhì)分子量為22.2211 KDa,PI為5.22,蛋白不穩(wěn)定系數(shù)為33.90。(2)軟件ExPASy、SignalP 3.0 Server、TMHMM Server V2.0和Prot Fun 2.2 Server分析結(jié)果顯示,基因OsHP29和基因OsHP40各編碼一個(gè)親水性蛋白,基因OsHP29編碼的蛋白屬于不含信號肽的非跨膜蛋白,該蛋白參與調(diào)控水稻生長發(fā)育、能量代謝過程;基因OsHP40編碼的蛋白屬于不含信號肽的非跨膜蛋白,是一個(gè)結(jié)構(gòu)蛋白,參與蛋白質(zhì)的結(jié)合和轉(zhuǎn)運(yùn)等過程。(3)過表達(dá)載體pCAMBIA1301-OsHP29和pCAMBIA1301-OsHP40通過農(nóng)桿菌介導(dǎo)的葉盤法轉(zhuǎn)化煙草,基因OsHP29獲得了T0代植株12株,經(jīng)分子檢測得到陽性植株6株,陽性率為50%;基因OsHP40獲得了T0代植株12株,經(jīng)分子檢測得到陽性植株7株,陽性率為58%。(4)陽性轉(zhuǎn)化植株和野生型植株的耐熱性試驗(yàn)數(shù)據(jù)顯示:高溫處理2小時(shí)結(jié)束時(shí),野生型植株葉片發(fā)生彎曲,葉片的葉長直線距離由高溫處理前的平均5.1 cm彎曲至高溫處理結(jié)束時(shí)的平均3.3 cm,差異達(dá)到極顯著水平;轉(zhuǎn)OsHP29基因的植株葉片也發(fā)生了一定的彎曲,葉片的葉長直線距離由高溫處理前的平均5.2 cm彎曲至高溫處理結(jié)束時(shí)的平均3.5 cm,差異達(dá)到顯著水平;而轉(zhuǎn)OsHP40基因的植株葉片受高溫影響較小,葉片的葉長直線在高溫處理前、結(jié)束時(shí)無顯著差異,表明目的基因OsHP29和OsHP40具有提高煙草耐熱性的功能。
[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.
【學(xué)位授予單位】：江西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S511
本文編號：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.
【學(xué)位授予單位】：江西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S511
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