【作者】 陈东亮；

【导师】 彭镇华；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2013， 博士

【摘要】 麻竹（Dendrocalamus latiflorus）是我国南方广泛栽培的重要竹种之一，具有食用、材用、绿化等多种用途，经济价值极高。植物基因的表达受到转录前、转录以及翻译水平等多个层的精准调控，研究竹子基因表达调控的机理对于深入了解竹子的生命现象，更好地开发利用竹子资源具有重要价值。转录因子参与基因转录水平的调控，miRNA参与转录后调控，二者都是植物基因表达调控的重要方式。本研究以麻竹为对象，针对SCL6和AP2这两个植物特有的转录因子进行了研究，同时开展了麻竹叶片miRNA的分析研究，并对其部分新miRNA的表达进行了分析探讨。主要研究成果如下：（1）利用RT-PCR和RACE方法，从麻竹叶片中克隆到与其生长发育密切相关的转录因子基因DlSCL6和DlAP2。DlSCL6基因cDNA全长2006bp，5′非编码区（UntranslatedRegions，UTR）124bp，3′UTR266bp，含有一个1611bp的开放阅读框（Open ReadingFrame，ORF），编码536个氨基酸；DlAP2基因cDNA全长1731bp，其中5′UTR81bp，3′UTR186bp，ORF1464bp，共编码487个氨基酸。（2）分别构建了DlSCL6基因的正义和反义表达载体，利用农杆菌介导法转化模式植物拟南芥（Arabidopsis thaliana），并对抗性植物进行RT-PCR鉴定，分别获得了DlSCL6基因的正义、反义转基因株系。对转基因植株表型观察显示，转正义DlSCL6基因植株营养期延长，开花延迟，植株粗壮高大，莲座叶数量增加；而转入反义基因的植株开花提前，植株瘦弱，且莲座叶数量明显减少。转反义DlSCL6基因植株表型与矮牵牛（Petuniahybrida）、拟南芥中ham基因功能缺失的表型类似，这意味着DlSCL6基因与拟南芥等植物中HAM亚家族基因具有类似功能，可能在竹子维持顶端分生组织分生活性中发挥重要作用。（3）将DlAP2基因构建到pCAMBIA1300载体的多克隆位点，形成DlAP2基因的过量表达载体，利用农杆菌介导法成功获得了转DlAP2基因的拟南芥植株，并利用RT-PCR方法证明DlAP2基因在转基因植株中稳定表达。与野生型拟南芥相比，转DlAP2基因植株开花提前，表明DlAP2可能具有促进麻竹开花的功能。（4）构建了麻竹叶片miRNA文库，利用Solexa高通量测序技术测序得到11513607条序列，去除低质量序列、5′端污染、3′端接头缺失、插入片段缺失、polyA片段以及小于18nt的序列后，得到干净序列（clean reads）10593305条。应用生物信息学分析技术预测得到165种候选的miRNA，其中与已知的其他物种miRNA同源性较高的保守miRNA有84种（其中miRNA54种，miRNA*30种），分别属于17个已知的miRNA家族；非保守的新miRNA共81种（其中miRNA76种，miRNA*5种）。搜索比对麻竹EST数据库和毛竹CDS数据库，对麻竹新miRNA的靶基因进行预测，得到靶基因176个，其中37个来自麻竹EST数据库，139个来自毛竹基因组CDS数据。（5）根据获得麻竹叶片新miRNA序列特征，设计高度特异的反转录茎环引物，对表达量较高的30种新miRNA进行了qRT-PCR检测。结果表明，所检测miRNA的溶解曲线均高度特异，表明其在麻竹叶片中是真实存在的，并非背景信号。采用qRT-PCR方法对这30种新miRNA在不同光照环境下的表达差异分析表明：在强光胁迫下（1200μmol/m2/s1）， dla-miRC18、 dla-miRC27、 dla-miRC27-3p的表达明显上调，其中dla-miRC27-5p的上调幅度最大（达到对照的15倍），而dla-miRC1、dla-miRC19和dla-miRC28的表达明显下调（62%-76%）；黑暗条件下，这些miRNA的表达变化相对较小，仅有dla-miRC1和dla-miR22的上调幅度比较明显（是对照的4倍左右），dla-miRC5、dla-miRC17和dla-miRC29的下调幅度达到50%-72%。由此表明，在麻竹的光信号转导、光胁迫反应等相关基因的表达中，这些miRNA可能发挥着重要调节作用。麻竹DlSCL6和DlAP2基因在模式植物拟南芥中的表达表明：这两个基因均对植物从营养生长向生殖生长的转换过程具有调控作用，为进一步深入研究竹子开花机理奠定了基础。对麻竹叶片miRNA的分离鉴定，获得了81条麻新的miRNA，并对部分新miRNA在不同光环境下的表达差异进行了研究，为进一步研究竹子miRNA功能奠定了基础。更多还原

【Abstract】 Ma bamboo (Dendrocalamus latiflorus) is one of the most widely cultivated bamboospecies with high economic value in the south of China, which has a variety of uses for food,timber and afforestation. The regulation of gene expression in plant precisely occurred at thelevels of transcription and translation. Research on bamboo mechanism of gene expressionregulation is of great value for deeper understanding of the phenomenon of bamboo life andbetter development and utilization of bamboo resources. Transcription factor (TF) andmiRNAs, which involved in the gene regulation at transcription and post-transcription levelsrespectively, are two important ways of controlling gene expression in plants. In this paper,two plant-specific transcription factors (SCL6and AP2) from ma bamboo were studied. At thesame time, isolation and identification of miRNAs from leaves of ma bamboo were carried outthrough deep sequencing, the expression of some novel miRNAs were further analyzed byusing qRT-PCR. The main research results are as follows:(1) Two plant-specific TF genes DlSCL6and DlAP2were cloned from ma bamboo leavesusing RT-PCR and RACE methods. The cDNA of DlSCL6is2006bp in full length including124bp in5’ UTR,266bp in3’ UTR, and an ORF of1611bp which encodes536amino acids.The cDNA of DlAP2is1731bp in full length and consists of a5’ UTR of81bp, a3’ UTR of186bp and an ORF of1464bp which encodes487amino acids.(2) Expression vectors of sense and antisense DlSCl6gene were constructed, and transformedinto Arabidopsis thaliana mediated by Agrobacterium tumefaciens respectively. Then thetransgenic plants were identified by RT-PCR method. Phenotype observation showed: thattransgenics plants with sense DlSCL6gene had prolonged vegetative growth period, delayedblooming, stronger stem, and more rosette leaves, while the transgenics of antisense DlSCL6geneshowed opposite phenotypes: such as ealier precocious flowering, weaker stem and less rosetteleaves. All of these phenotypes are similar to the phenotypes of ham deficient mutant in A.thalianaand petunia. All of this results indicated that DlSCL6has similar function with HAM genes in A.thaliana and petunia, and maybe play important roles in the maintenance of bamboo stem apicalmeristem (SAM).(3) Overexpression vector of DlAP2gene was constructed, and transformed intoA.thaliana by Agrobacterium-mediated method. The transgenic plants were selected byHygromycin B, and DlAP2was proved expression at transcription level by RT-PCR method.The transgenic plants bloomed earlier than that of wild type, which indicated that DlAP2maysplays a role as blooming promoter.(4) The miRNA library was constructed with leaves of ma bamboo using Illumina kit.11513607raw reads were obtained using Solexa high-throughput sequencing technology. Afterremoving the sequences of low quality,5’end of pollution,3’ end joint deletion, insertion,polyA and less than18nt sequence,10593305clean reads was left. Bioinformatics methodswere used to forecast possible miRNAs, and total165candidate miRNA was identified, whichincuded84conversed miRNAs (54mature miRNAs and30star miRNAs) belongs to17converse miRNA families and84novel miRNAs (76mature miRNAs and5star miRNAs).Based on two data sets of CDS for moso genome and ESTs for ma bamboo,176potentialtargets for81novel miRNAs were forecasted.(5) Highly specific stem-loop reverse primers were designed according to the miRNAssequences, and30higher expression novel miRNAs were detected by qRT-PCR methods.Highly specific dissolution curves indicated that these miRNAs were really exist in ma bamboo.Further analysis for the expression of these miRNAs in leaves under different light conditionsshowed that dla-miRC18, dla-miRC27and dla-miRC27-3p were upregulated significantlyunder high light tress (1200μmol/m2/s1), in which dla-miRC18was the hightest one with up to15times compared to that of control, whilt dla-miRC1, dla-miRC19and dla-miRC28weredownregulated, decreased by62%-76%. Under dark conditions for24h, only dla-miRC1anddla-miR22were significantly upregulated, about4times of the control, contrarily dla-miRC5,dla-miRC17and dla-miRC29downregulated significantly (decreased by50%-72%). All theresults above indicated that these miRNAs might play important roles in genes regulationinvolved in light signal transduction and light stress. The expression of DlSCL6and DlAP2in Arabidopsis thaliana showed that both DlSCL6and DlAP2gene play roles in the control of transition from vegetative growth to reproductivegrowth, which provides some reference for the mechanism study on bamboo blossom.81newmiRNAs were identificated from bamboo leaves, and the expression changes of30novelmiRNAs under different light conditons were studied, which lays a foundation for further studyon their functions.更多还原