NaCl处理对酸枣苗AsA-GSH循环的影响与miRNA靶基因降解组分析
发布时间：2018-09-19 19:48
【摘要】：本文以酸枣(Ziziphus acidojujuba C.Y.Cheng er M.J.Liu)水培苗为试材,探究了不同浓度NaCl(0、50、100、150、200 mmol?L-1)和CaCl2处理对酸枣幼苗根、茎、叶中AsA-GSH循环的影响,并成功构建了CK和NaCl处理2个酸枣叶片降解组文库,通过降解组测序鉴定已知miRNA和新miRNA的靶基因,为进一步研究酸枣响应盐胁迫的miRNA功能研究提供依据。利用qPCR检测并利用geNorm、NormFinder、BestKeeper软件评价了NaCl胁迫0、6、12、24和48 h酸枣幼苗叶片中8个miRNA候选内参基因5S rRNA、5.8S rRNA、U6 snRNA、miR156q、miR319、miR472a、novel_mir_50、novel_mir_62和5个mRNA候选内参基因Actin、EF-1α、GADPH、H3、UBQ的稳定性,筛选出适于检测NaCl胁迫下酸枣幼苗叶片中miRNA及其靶基因表达量的稳定内参。主要研究结果如下:1.与对照相比,随着NaCl处理浓度的升高,酸枣苗根、茎、叶中AsA含量均增加;DHA含量也呈上升趋势;叶中AsA/DHA比值呈先上升后下降的趋势;叶中GSH含量变化呈现先升高后下降的趋势;200 mmol?L-1 NaCl处理下茎中GSSG含量显著升高;不同浓度NaCl处理下,茎中GSH/GSSG比值呈先升高后下降的趋势;酸枣幼苗各器官中APX活性随着NaCl浓度的升高逐渐下降,GR、MDHAR活性与对照相比存在显著差异。营养液中加入10 mmol?L-1 CaCl2后,与NaCl处理相比,酸枣根和茎中AsA含量进一步升高,叶中AsA含量下降,APX活性下降,根中MDHAR活性进一步升高,茎和叶中DHAR活性升高;根、茎、叶中GSH含量下降。高浓度NaCl胁迫下,酸枣幼苗根、茎、叶中AsA、DHA含量增加,APX活性下降;茎和叶中GSSG含量增加。NaCl胁迫下,酸枣幼苗AsA-GSH循环中催化AsA再生的主要酶可能是MDHAR。外源CaCl2可能通过提高酸枣实生苗根、茎、叶中AsA再生关键酶MDHAR和DHAR活性,促进根、茎和叶中AsA的再生与生物合成,从而提高根、茎、叶中AsA含量起到清除过量H2O2缓解NaCl胁迫对酸枣幼苗根、茎和叶的伤害。2.利用qPCR和geNorm、NormFinder和BestKeeper软件检测并评价了NaCl处理0、6、12、24、48 h酸枣苗叶中8个miRNA(5S rRNA、5.8S rRNA、U6 snoRNA、miR472a、miR319、miR156q、novel_mir_50、novel_mir_62)及5个mRNA(GADPH、UBQ、H3、EF1-α、Actin)候选内参基因的表达稳定性。综合不同软件分析结果表明,NaCl胁迫不同时间点酸枣苗叶片中表达较稳定的miRNA候选内参基因是5S rRNA和miR156q,mRNA是Actin和H3。3.通过对CK和NaCl两个降解组文库的高通量测序,分别得到634,882,392和601,821,464条reads,分别有10,517,778(77.86%)和9,947,967(77.69%)条reads能够比对到枣(Ziziphus jujuba Mill.)基因组上。利用Rfam、GenBank等数据库对得到的序列进行分类注释,并对mRNA来源的片段进行miRNA降解位点鉴定。预测到已知和新miRNA的232个靶基因,其中57个为28个已知miRNA靶基因,175个为126个新miRNA的靶基因。功能分析显示,这些靶基因参与酸枣生长发育、转录调节、胁迫应答等方面,其中部分基因可能与酸枣响应NaCl胁迫相关。
[Abstract]:In this paper, the effects of different concentrations of NaCl (050100150200 mmol?L-1) and CaCl2 on AsA-GSH cycle in roots, stems and leaves of jujube seedlings were studied, and two biodegradable libraries of CK and NaCl were successfully constructed. The target genes of known miRNA and new miRNA were identified by sequencing the degradation group, which provided the basis for further study on the miRNA function of jujube in response to salt stress. The stability of 8 miRNA candidate internal reference genes 5S rRNA,5.8S rRNA,U6 snRNA,miR156q,miR319,miR472a,novel_mir_50,novel_mir_62 and 5 mRNA candidate internal reference genes Actin,EF-1 伪 GADPHH3H3UBQs in leaves of Ziziphus jujuba seedlings under NaCl stress at 24 and 48 h were detected by qPCR and evaluated by geNorm,NormFinder,BestKeeper software. A stable internal reference was selected to detect the expression of miRNA and its target genes in the leaves of jujube seedlings under NaCl stress. The main results are as follows: 1. Compared with the control, the content of AsA in roots, stems and leaves of jujube seedlings increased with the increase of NaCl concentration, and the AsA/DHA ratio in leaves increased first and then decreased. The content of GSH in leaves increased at first and then decreased. The content of GSSG in stem increased significantly under 200 mmol?L-1 NaCl treatment, and the ratio of GSH/GSSG in stem increased first and then decreased under different concentration of NaCl. The activity of APX in the organs of jujube seedlings decreased gradually with the increase of NaCl concentration, and the activity of GRMDHAR was significantly different from that of the control. Compared with NaCl treatment, the content of AsA in root and stem of Zizyphus jujuba was further increased, the content of AsA in leaf decreased, the activity of MDHAR in root increased further, the activity of DHAR in stem and leaf increased, the content of AsA in root and stem increased after adding 10 mmol?L-1 CaCl2 to the nutrient solution, and the content of AsA in root and stem of jujube increased. The content of GSH in leaves decreased. Under high concentration of NaCl stress, the content of AsA,DHA in roots, stems and leaves of jujube seedlings increased and decreased. Under the stress of GSSG content in stems and leaves, the main enzyme to catalyze the regeneration of AsA in AsA-GSH cycle of jujube seedlings may be MDHAR.. Exogenous CaCl2 may promote the regeneration and biosynthesis of AsA in roots, stems and leaves of jujube seedlings by increasing the activities of MDHAR and DHAR, the key enzymes of AsA regeneration in roots, stems and leaves, thus increasing the regeneration and biosynthesis of AsA in roots, stems and leaves. The content of AsA in leaves can eliminate excessive H2O2 and alleviate the damage of NaCl stress to roots, stems and leaves of jujube seedlings. 2. QPCR, geNorm,NormFinder and BestKeeper software were used to detect and evaluate the expression stability of 8 miRNA (5S rRNA,5.8S rRNA,U6 snoRNA,miR472a,miR319,miR156q,novel_mir_50,novel_mir_62) and 5 mRNA (GADPH,UBQ,H3,EF1- 伪 -actin) candidate internal reference genes in leaves of Zizyphus jujuba seedlings treated with NaCl. The results of different software analysis showed that 5s rRNA and miR156q,mRNA were 5s rRNA and miR156q,mRNA Actin and H3.3 genes in leaves of Zizyphus jujuba seedlings at different time points. Through the high-throughput sequencing of CK and NaCl degradation group library, 10517778 (77.86%) and 9947967 (77.69%) reads of 634882392 and 601821464 reads, were obtained respectively, which can be compared to (Ziziphus jujuba Mill.) of jujube. On the genome. The sequence was classified and annotated by Rfam,GenBank and mRNA fragments were identified by miRNA degradation site. 232 target genes of known and new miRNA were predicted, of which 57 were 28 known miRNA target genes and 175 were new miRNA target genes. Functional analysis showed that these target genes were involved in the growth and development of jujube, transcription regulation and stress response, and some of them might be related to the response of Zizyphus jujuba to NaCl stress.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S665.1
本文编号：2251148
[Abstract]:In this paper, the effects of different concentrations of NaCl (050100150200 mmol?L-1) and CaCl2 on AsA-GSH cycle in roots, stems and leaves of jujube seedlings were studied, and two biodegradable libraries of CK and NaCl were successfully constructed. The target genes of known miRNA and new miRNA were identified by sequencing the degradation group, which provided the basis for further study on the miRNA function of jujube in response to salt stress. The stability of 8 miRNA candidate internal reference genes 5S rRNA,5.8S rRNA,U6 snRNA,miR156q,miR319,miR472a,novel_mir_50,novel_mir_62 and 5 mRNA candidate internal reference genes Actin,EF-1 伪 GADPHH3H3UBQs in leaves of Ziziphus jujuba seedlings under NaCl stress at 24 and 48 h were detected by qPCR and evaluated by geNorm,NormFinder,BestKeeper software. A stable internal reference was selected to detect the expression of miRNA and its target genes in the leaves of jujube seedlings under NaCl stress. The main results are as follows: 1. Compared with the control, the content of AsA in roots, stems and leaves of jujube seedlings increased with the increase of NaCl concentration, and the AsA/DHA ratio in leaves increased first and then decreased. The content of GSH in leaves increased at first and then decreased. The content of GSSG in stem increased significantly under 200 mmol?L-1 NaCl treatment, and the ratio of GSH/GSSG in stem increased first and then decreased under different concentration of NaCl. The activity of APX in the organs of jujube seedlings decreased gradually with the increase of NaCl concentration, and the activity of GRMDHAR was significantly different from that of the control. Compared with NaCl treatment, the content of AsA in root and stem of Zizyphus jujuba was further increased, the content of AsA in leaf decreased, the activity of MDHAR in root increased further, the activity of DHAR in stem and leaf increased, the content of AsA in root and stem increased after adding 10 mmol?L-1 CaCl2 to the nutrient solution, and the content of AsA in root and stem of jujube increased. The content of GSH in leaves decreased. Under high concentration of NaCl stress, the content of AsA,DHA in roots, stems and leaves of jujube seedlings increased and decreased. Under the stress of GSSG content in stems and leaves, the main enzyme to catalyze the regeneration of AsA in AsA-GSH cycle of jujube seedlings may be MDHAR.. Exogenous CaCl2 may promote the regeneration and biosynthesis of AsA in roots, stems and leaves of jujube seedlings by increasing the activities of MDHAR and DHAR, the key enzymes of AsA regeneration in roots, stems and leaves, thus increasing the regeneration and biosynthesis of AsA in roots, stems and leaves. The content of AsA in leaves can eliminate excessive H2O2 and alleviate the damage of NaCl stress to roots, stems and leaves of jujube seedlings. 2. QPCR, geNorm,NormFinder and BestKeeper software were used to detect and evaluate the expression stability of 8 miRNA (5S rRNA,5.8S rRNA,U6 snoRNA,miR472a,miR319,miR156q,novel_mir_50,novel_mir_62) and 5 mRNA (GADPH,UBQ,H3,EF1- 伪 -actin) candidate internal reference genes in leaves of Zizyphus jujuba seedlings treated with NaCl. The results of different software analysis showed that 5s rRNA and miR156q,mRNA were 5s rRNA and miR156q,mRNA Actin and H3.3 genes in leaves of Zizyphus jujuba seedlings at different time points. Through the high-throughput sequencing of CK and NaCl degradation group library, 10517778 (77.86%) and 9947967 (77.69%) reads of 634882392 and 601821464 reads, were obtained respectively, which can be compared to (Ziziphus jujuba Mill.) of jujube. On the genome. The sequence was classified and annotated by Rfam,GenBank and mRNA fragments were identified by miRNA degradation site. 232 target genes of known and new miRNA were predicted, of which 57 were 28 known miRNA target genes and 175 were new miRNA target genes. Functional analysis showed that these target genes were involved in the growth and development of jujube, transcription regulation and stress response, and some of them might be related to the response of Zizyphus jujuba to NaCl stress.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S665.1
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