【作者】 江舟；

【导师】 潘光堂； 沈亚欧；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2013， 博士

【摘要】 传统的转基因玉米新品种培育需经遗传转化、自交纯合、杂交组配及回交转育几个重要环节,通常从转化体自交纯合到回交转育获得转基因骨干自交系至少需要3～6年。作为世界玉米转基因育种最广泛使用的外植体——玉米胚性愈伤组织,其诱导率受基因型限制。就我国转基因玉米育种工作而言,大多数玉米骨干系胚性愈伤组织诱导率低,不能直接作为转基因受体材料,因而严重阻碍了我国转基因玉米育种的快速发展。miRNA是一类重要的内源性非编码RNA,它通过与靶mRNA序列部分或者完全匹配从而行使对基因的剪切、抑制翻译等,对其表达进行调控,广泛参与植物生长发育及逆境胁迫响应过程；玉米转基因育种的理论研究与无数实践均已经证明,玉米胚性愈伤组织诱导率受基因型限制。因此,本研究通过miRNA途径探讨玉米胚性愈伤组织发生率的遗传机理,以期培育更多胚性愈伤组织高诱导率的骨干亲本系,提高我国玉米基因工程育种的效率。本研究选用玉米胚性愈伤组织强诱导能力自交系18-599R为供试材料,利用solexa高通量测序技术与生物信息学分析方法,挖掘获得了大量18-599R未成熟胚经2,4-D诱导形成胚性愈伤组织过程相关的miRNA,结合荧光定量PCR和miRNA转基因过表达技术,对重要候选miRNA进行了表达检测及基因功能初步验证；并通过靶基因降解组高通量测序技术获得了胚性愈伤组织形成过程相关miRNA的靶基因信息。通过本研究,获得以下研究结果与结论：(1) solexa高通量测序共筛选获得了20个已知miRNA保守家族的78个差异表达miRNA。从对照(Control)到愈伤诱导过程的后3阶段(Stage Ⅰ、StageⅡ和StageⅢ),共有45个miRNA呈上调表达趋势,占差异表达miRNA的57.69%；下调表达的miRNA占29.49%,共计23个；另外有10个miRNA在愈伤诱导全过程中表达有上调也有下调,占12.82%。表明大多数：miRNA均以上调表达趋势参与了玉米胚性愈伤组织形成过程的调控。(2)本研究发现了13个miRNA与zma-miR167、zma-miR172、zma-miR319、 zma-miR393、zma-miR396、zma-miR397、zma-miR408和zma-miR528等8个已知的玉米miRNA家族成员序列保守性极高,经进一步生物信息学分析证实它们是这些miRNA家族的新成员。(3)通过高通量测序挖掘获得7个新miRNA,属于4个miRNA家族,分别命名为：zma-miR701、zma-miR702、zma-miR703和zma-miR704,它们在愈伤形成过程存在不同程度及类型的差异表达。这些新miRNA长度为21-22nt,位于其二级结构3’端茎部,并且均具有较低的折叠自由能和典型的二级结构。(4)通过聚类分析可将测序获得的已知家族差异表达的miRNA分为4类表达模式：下调模式、表达差异不显著模式、上调模式和显著上调模式。同样表明参与调控胚性愈伤组织形成过程的miRNA多数为上调表达模式。对这4类miRNA表达模式的进一步分析推测,Stage II(初级愈伤形成期)可能是胚性愈伤组织形成过程中重要的质变阶段,与玉米胚性愈伤组织的形成密切相关。(5)通过荧光定量PCR验证了测序结果中14个差异表达的miRNA,发现除zma-miR156a、zma-miR167a、zma-miR1671和zma-miR827等4个miRNA下调表达,其余10个均显著上调表达。另外,定量PCR验证结果同部分测序结果的相关系数达到0.94以上,证明本研究测序结果准确性较高。(6)通过降解组测序获得了213个靶基因,分别受22个已知miRNA保守家族介导剪切。这些靶基因可分为细胞组成、分子功能和生物过程三类,分别参与调节植物生长发育、转录调控、胁迫响应、激素信号传导、植物代谢途径等过程。其靶基因功能主要涉及SBP、TCP、GAMYB、ARF、F-box和GRAS等转录因子。(7)降解组结果经KO分析,发现与胚性愈伤组织形成过程密切相关的显著通路是激素信号转导途径。愈伤形成过程中共有14个靶基因在该通路中表达,分别受zma-miR160、zma-miR167、zma-miR393和zma-miR394等4个]miRNA负调控,从而调控激素信号的转导,影响内源激素合成,最终调控胚性愈伤组织的形成。(8)对转基因T1代阳性植株幼胚胚性愈伤组织诱导率的鉴定结果表明,过表达zma-miR159a和zma-miR393a的转基因植株诱导率显著或极显著低于野生型对照,证明这些miRNA确实参与调控胚性愈伤组织形成过程。更多还原

【Abstract】 Creating new germplasm resources by transgenic methods is a fundamental way to broaden the maize germplasm resources which is relatively strait in China. The conventional way of cultivating new transgenic variety of maize consists of several critical steps including genetic transformation, self-crossing for homozygosis, crossing combination, and back crossing, among which the process from self-crossing transformants to acquiring the transgenic elite inbred lines by backcrossing usually takes at least3to6years.The infeasibility of using embryonic callus as the transgenic recipient material directly, which is attributed to the low frequency of maize embryonic callus introduction being affected by genotype significantly in the majority of elite inbred lines of maize, severely hinders the rapid development of breeding for transgenic maize in China. By regulating plant gene expression through ways such as directing gene splicing or suppressing transcription, miRNA, a class of endogenous non-coding RNA, involves widely in every stage of plant growth and development, as well as responds to the adversity stress.In this study, the maize inbred lines18-599R with the strong ability of embryonic culturing were used as test materials, many candidate miRNAs involved in the process of embryonic callus formation in immature embryos of18-599R under the2,4-D induction were screened based on the initial research combining Solexa high-throughput sequencing with bioinformatics analysis methods; by combing the RT-PCR with miRNA overexpression technology, expression of important candidate miRNAs were detected and the function of target genes were verified; information of target genes of miRNA involved in the formation of embryonic callus was acquired through the high-throughput Degradome Sequencing for target genes. And the final results are as follows;1.78differentially expressed miRNAs from25known conservative miRNA families were screened out by Solexa high-throughput sequencing. From the control to the last3stages (Stage Ⅰ,Stage Ⅱ and Stage Ⅲ) of callus induction,45miRNAs accounting for the57.69%of differentially expressed miRNAs were up-regulated, while23miRNAs accounting for the29.49%of total were down-regulated; in addition,10miRNAs accounting for the12.82%of all were up-regulated as well as down regulated. 2.13miRNAs detected in this study were of significant sequence conservation with members of8known maize miRNA families including zma-miR167、zma-miR172、 zma-miR319、zma-miR393、zma-miR396、zma-miR397、zma-miR408and zma-miR528, the further bioinformatics analysis confirmed that they are novel members of those miRNA families.3.7new miRNAs belonging to4miRNA families named zma-miR701, zma-miR702, zma-miR703, zma-miR704, respectively, with various levels and types of differentially expression during the formation of callus, were identified by high-throughput sequencing. Those new miRNAs with the length ranging from21to22nt and3’in the stem of secondary structure, are of relatively low refolding free energy and emblematical secondary structure.4. According to the cluster analysis, expression models of those sequencing-detected differentially expressed miRNAs of know families were categorized into four groups including down-regulating model, inconspicuously differential expression model, up-regulating model, significantly up-regulating model. Based on the further analysis for the4expression models, we presume that Stage II(the stage of the formation of primary callus) may be the most important stage during the formation of embryonic callus, and closely related to the formation of embryonic callus of maize.5.14of the differentially expressed miRNAs detected by sequencing were verified by RT-PCR, showing that10were significantly up-regulated while the rest4zma-miR156a, zma-miR167a, zma-miR1671and zma-miR827were down-regulated. In addition, the correlation coefficient of the results of qRT-PCR verification and sequencing is above0.94, indicating the high accuracy of sequencing results in this study.6.213target genes sliced under the induction of22known miRNA conservative families were identified by the degradome sequencing. These target genes involved in the regulation of processes including plant growth and development, transcriptional regulation, stress responses, hormone signaling, plant metabolic pathways can be categorized into three types:cell composition, molecular function and biological process. Their target gene functions involve transcription factors including SBP, TCP, GAMYB, ARF, F-box and GRAS.7. The hormone signaling pathway was identified being significantly correlated with the formation of embryonic callus by KO analysis for the results of degradome sequencing. 14target genes were differentially expressed in this pathway under the negative regulations of4miRNAs including zma-miR160, zma-miR167, zma-miR393, zma-miR394, respectively.8. According to the phenotype identification for the embryonic callus induction rate of immature embryo of T1generation transgenic positive plants, the induction rate of transgenic plants overexpressing zma-miR159a or zma-miR393a is significantly or extremely significantly lower than that of wild-type controls. There is a difference between the results and the expected transgene results更多还原