【作者】 李超汉；

【导师】 于贤昌；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2014， 博士

【摘要】 黄瓜（Cucumis sativus）与南瓜（Cucurbita moschata）砧木嫁接能明显促进黄瓜生长，提高黄瓜抗逆性。植物microRNA（miRNA）在转录后抑制靶基因表达，调节植物生长发育和应答各种非生物胁迫。然而尚缺乏miRNA调控黄瓜嫁接苗生长和抗逆性的研究。为研究miRNA参与调控黄瓜嫁接苗生长发育和抵御非生物胁迫的作用机制，分别以①黄瓜—南瓜（接穗—砧木）嫁接的黄瓜嫁接苗、②南瓜—黄瓜（接穗—砧木）嫁接的南瓜嫁接苗、③黄瓜自根嫁接苗和④南瓜自根嫁接苗的叶片和根尖构建了8个小RNA文库。利用高通量测序与生物信息学手段，鉴定黄瓜与南瓜中响应嫁接的miRNA。对黄瓜嫁接苗和黄瓜自根嫁接苗进行干旱、高盐、缺氮或缺磷胁迫，利用荧光定量PCR检测叶片和根中miRNA和靶基因的表达。主要结果如下：1.鉴定黄瓜和南瓜miRNA。利用Solexa测序，结合BLASTn和Mireap等，鉴定到112条已知miRNA（分属40个miRNA家族）以及48条新miRNA，预测了miRNA的靶基因。2.鉴定黄瓜中响应嫁接的miRNA。高通量测序结果显示，黄瓜嫁接苗与南瓜嫁接苗中75%的已知miRNA的表达发生显著变化。荧光定量PCR发现10条已知miRNA、16条新miRNA以及19条靶基因的表达均发生了显著变化，说明miRNA及其靶基因能响应嫁接。3. miRNA参与黄瓜嫁接苗应答干旱胁迫。与黄瓜自根嫁接苗相比，黄瓜嫁接苗叶片中多数miRNA表达在干旱3h后下调，8h和24h后明显上调；根中多数miRNA表达在干旱1h后下调，3h和8h后明显上调，24h后下调；叶片中多数靶基因表达在干旱3h后明显上调，8h和24h后下调；根中多数靶基因表达在干旱胁迫下明显上调。4. miRNA参与黄瓜嫁接苗应答盐胁迫。与黄瓜自根嫁接苗相比，黄瓜嫁接苗叶片多数miRNA表达在盐胁迫2h后下调，6h和24h后上调；根中多数miRNA表达在盐胁迫下明显上调；叶片中多数靶基因表达在盐胁迫24h后明显下调；根中多数靶基因表达在盐胁迫下明显上调。5. miRNA参与黄瓜嫁接苗应答缺氮或缺磷胁迫。与黄瓜自根嫁接苗相比，黄瓜嫁接苗叶片多数miRNA表达在缺氮或缺磷1h后明显下调，3h后上调，8h后又下调，缺氮24h和48h后上调，而缺磷24h和48h后明显下调；根中多数miRNA表达在缺氮24h后明显上调；缺磷1h和3h后上调，8h和24h后下调，48h后上调；叶片中多数靶基因表达在缺氮1h、3h和8h后明显下调，24h和48h后上调；缺磷1h和3h后明显上调，8h、24h和48h后下调；根中多数靶基因表达在缺氮或缺磷胁迫下明显上调。本研究为阐明miRNA参与调控黄瓜嫁接苗生长发育和抵御非生物胁迫的作用机制奠定了理论基础。更多还原

【Abstract】 Pumpkins (Cucurbita moschata) are usually used as rootstocks for cucumber (Cucumis sativus)grafting to improve cucumber growth and tolerance to abiotic stresses. Plant microRNAs (miRNAs) areinvolved in regulating plant growth and development, and response to various abiotic stresses throughcleavage of target genes at post-transcriptional level. However, the miRNAs-mediated regulation ongrafting-induced enhancement of growth and stress tolerances in cucumber still needs further study.With the aim of probing the molecular mechanism of miRNAs-mediated growth and resistance toabiotic stresses of grafted cucumber seedlings, we constructed eight small RNA libraries from leavesand roots of seedlings that were grafted in the following four ways:(1) hetero-grafted cucumberseedling, using cucumber as scion and pumpkin as rootstock;(2) hetero-grafted pumpkin seedling, usingpumpkin as scion and cucumber as rootstock;(3) auto-grafted cucumber seedling, and (4) auto-graftedpumpkin seedling. By high-throughput sequencing and bioinformatics analysis, a group ofgrafting-responsive miRNAs were identified. And after treatments of drought, high salinity, nitrogen orphosphorus deficiencies, the expressions of miRNAs and target mRNAs in hetero-grafted cucumberseedlings and auto-grafted cucumber seedlings were detected by quantitative real-time PCR (qRT-PCR).The main results are as follows:1. Identification of miRNAs in cucumber and pumpkin. By Solexa high-throughput sequencing andbioinformatics analysis such as BLASTn and Mireap,112known miRNAs belonging to40miRNAfamilies, and48novel miRNAs were identified. The target genes of these miRNAs were predicted.2. Identification of grafting-responsive miRNAs in cucumber. According to the sequencing data,compared with auto-grafted seedlings, expressions of75%known miRNAs were significantly changed.The expressions of10known and16novel miRNA as well19predicted target genes were detected byqRT-PCR, and the expressions of miRNAs and target genes were changed dynamically in leaves androots of hetero-grafted cucumber seedlings. The expression-changed miRNAs and target genes wereconsidered as grafting-responsive miRNAs and targets.3. miRNAs of grafted cucumber seedlings are involved in response to drought stress. Underdrought stress, compared with auto-grafted cucumber seedlings, expressions of most miRNAs in leavesof hetero-grafted cucumber seedlings were dramatically reduced after3h, and then induced after8hand24h of drought stress; expressions of most miRNAs in roots of hetero-grafted cucumber seedlingswere reduced after1h, while induced after3h and8h, and then reduced significantly after24h ofdrought stress. On the other hand, compared with auto-grafted cucumber seedlings, expressions of mosttargets in leaves of hetero-grafted cucumber seedlings were remarkably induced after3h, and thenreduced after8h and24h of drought stress; expressions of most targets in roots of hetero-graftedcucumber seedlings were significantly induced under drought stress.4. miRNAs of grafted cucumber seedlings are involved in response to salt stress. Under salt stress,compared with auto-grafted cucumber seedlings, expressions of miRNAs in leaves of hetero-grafted cucumber seedlings were significantly reduced after2h, while induced after6h and24h of salt stress;expressions of most miRNAs in roots of hetero-grafted cucumber seedlings were induced under saltstress. On the other hand, compared with auto-grafted cucumber seedlings, expressions of most targetgenes in leaves of hetero-grafted cucumber seedlings were reduced after2h, and then dramaticallyreduced after24h of salt stress; most target genes showed induced expressions in roots of hetero-graftedcucumber seedlings under salt stress.5. miRNAs of grafted cucumber seedlings are involved in response to nitrogen (N) or phosphorus(P) deficiencies. Compared with auto-grafted cucumber seedlings, expressions of most miRNAs inleaves of hetero-grafted cucumber seedlings were significantly reduced after1h of N or P deficiencies,and induced after3h of N or P deficiencies, and then reduced after8h of N or P deficiencies, andinduced after24h and48h of N deficiency, while reduced after24h and48h of P deficiency;expressions of most miRNAs in roots of hetero-grafted cucumber seedlings were induced remarkablyafter24h of N deficiency, while induced after3h of P deficiency, and reduced after8h and24h, andthen induced after48h of P deficiency. On the other hand, compared with auto-grafted cucumberseedlings, expressions of most target genes in leaves of hetero-grafted cucumber seedlings were reducedafter1h,3h and8h, and then induced after24h and48h of N deficiency, while induced after1h and3h, and then reduced after8h,24h and48h of P deficiency; most target genes in roots ofhetero-grafted cucumber seedlings showed induced expressions under N or P deficiencies.Our results are helpful for clarifying mechanisms of the miRNAs-mediated roles in improvinggrowth and abiotic stresses tolerance of grafted cucumber seedlings.更多还原