NaCl處理對(duì)酸棗苗AsA-GSH循環(huán)的影響與miRNA靶基因降解組分析
發(fā)布時(shí)間：2018-09-19 19:48
【摘要】：本文以酸棗(Ziziphus acidojujuba C.Y.Cheng er M.J.Liu)水培苗為試材,探究了不同濃度NaCl(0、50、100、150、200 mmol?L-1)和CaCl2處理對(duì)酸棗幼苗根、莖、葉中AsA-GSH循環(huán)的影響,并成功構(gòu)建了CK和NaCl處理2個(gè)酸棗葉片降解組文庫(kù),通過(guò)降解組測(cè)序鑒定已知miRNA和新miRNA的靶基因,為進(jìn)一步研究酸棗響應(yīng)鹽脅迫的miRNA功能研究提供依據(jù)。利用qPCR檢測(cè)并利用geNorm、NormFinder、BestKeeper軟件評(píng)價(jià)了NaCl脅迫0、6、12、24和48 h酸棗幼苗葉片中8個(gè)miRNA候選內(nèi)參基因5S rRNA、5.8S rRNA、U6 snRNA、miR156q、miR319、miR472a、novel_mir_50、novel_mir_62和5個(gè)mRNA候選內(nèi)參基因Actin、EF-1α、GADPH、H3、UBQ的穩(wěn)定性,篩選出適于檢測(cè)NaCl脅迫下酸棗幼苗葉片中miRNA及其靶基因表達(dá)量的穩(wěn)定內(nèi)參。主要研究結(jié)果如下:1.與對(duì)照相比,隨著NaCl處理濃度的升高,酸棗苗根、莖、葉中AsA含量均增加;DHA含量也呈上升趨勢(shì);葉中AsA/DHA比值呈先上升后下降的趨勢(shì);葉中GSH含量變化呈現(xiàn)先升高后下降的趨勢(shì);200 mmol?L-1 NaCl處理下莖中GSSG含量顯著升高;不同濃度NaCl處理下,莖中GSH/GSSG比值呈先升高后下降的趨勢(shì);酸棗幼苗各器官中APX活性隨著NaCl濃度的升高逐漸下降,GR、MDHAR活性與對(duì)照相比存在顯著差異。營(yíng)養(yǎng)液中加入10 mmol?L-1 CaCl2后,與NaCl處理相比,酸棗根和莖中AsA含量進(jìn)一步升高,葉中AsA含量下降,APX活性下降,根中MDHAR活性進(jìn)一步升高,莖和葉中DHAR活性升高;根、莖、葉中GSH含量下降。高濃度NaCl脅迫下,酸棗幼苗根、莖、葉中AsA、DHA含量增加,APX活性下降;莖和葉中GSSG含量增加。NaCl脅迫下,酸棗幼苗AsA-GSH循環(huán)中催化AsA再生的主要酶可能是MDHAR。外源CaCl2可能通過(guò)提高酸棗實(shí)生苗根、莖、葉中AsA再生關(guān)鍵酶MDHAR和DHAR活性,促進(jìn)根、莖和葉中AsA的再生與生物合成,從而提高根、莖、葉中AsA含量起到清除過(guò)量H2O2緩解NaCl脅迫對(duì)酸棗幼苗根、莖和葉的傷害。2.利用qPCR和geNorm、NormFinder和BestKeeper軟件檢測(cè)并評(píng)價(jià)了NaCl處理0、6、12、24、48 h酸棗苗葉中8個(gè)miRNA(5S rRNA、5.8S rRNA、U6 snoRNA、miR472a、miR319、miR156q、novel_mir_50、novel_mir_62)及5個(gè)mRNA(GADPH、UBQ、H3、EF1-α、Actin)候選內(nèi)參基因的表達(dá)穩(wěn)定性。綜合不同軟件分析結(jié)果表明,NaCl脅迫不同時(shí)間點(diǎn)酸棗苗葉片中表達(dá)較穩(wěn)定的miRNA候選內(nèi)參基因是5S rRNA和miR156q,mRNA是Actin和H3。3.通過(guò)對(duì)CK和NaCl兩個(gè)降解組文庫(kù)的高通量測(cè)序,分別得到634,882,392和601,821,464條reads,分別有10,517,778(77.86%)和9,947,967(77.69%)條reads能夠比對(duì)到棗(Ziziphus jujuba Mill.)基因組上。利用Rfam、GenBank等數(shù)據(jù)庫(kù)對(duì)得到的序列進(jìn)行分類注釋,并對(duì)mRNA來(lái)源的片段進(jìn)行miRNA降解位點(diǎn)鑒定。預(yù)測(cè)到已知和新miRNA的232個(gè)靶基因,其中57個(gè)為28個(gè)已知miRNA靶基因,175個(gè)為126個(gè)新miRNA的靶基因。功能分析顯示,這些靶基因參與酸棗生長(zhǎng)發(fā)育、轉(zhuǎn)錄調(diào)節(jié)、脅迫應(yīng)答等方面,其中部分基因可能與酸棗響應(yīng)NaCl脅迫相關(guān)。
[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 偽 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- 偽 -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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S665.1
本文編號(hào)：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 偽 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- 偽 -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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S665.1
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