【作者】 陈晓；

【导师】 陈彦惠；

【作者基本信息】 河南农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 热带、亚热带玉米种质具有丰富的遗传变异,是拓宽我国玉米种质遗传基础和提升育种水平的一个重要途径。然而,玉米光周期敏感是热带、亚热带种质利用的一个主要障碍。因此,分离、克隆控制玉米光周期敏感相关基因并探知其功能,进而揭示光周期敏感的遗传特性和内在机制,不仅对热带、亚热带玉米种质的有效利用,而且对植物花发育生物学机理研究的深入,均具有重要的理论和实践意义。本研究以热带玉米光周期敏感自交系CML288为材料,通过长、短日照挪移试验,在明确玉米对光周期反应敏感指标和敏感时期的基础上,利用抑制差减杂交的方法,构建了具有3000多个单克隆的光周期敏感差减文库;利用同源基因克隆结合RACE技术,分离克隆了ZmHd1、PL5L15和PL3K2等同玉米光周期敏感反应相关的重要基因,并分析了克隆基因在不同发育时期和器官的表达特点;然后根据生物信息学分析,通过与RIL群体光周期敏感基因QTL定位研究结果的比较,初步对ZmHd1基因进行了定位和功能的验证。主要研究结果如下:1、通过对CML288和黄早四玉米自交系光周期敏感特性对比试验发现,在9小时短日照条件下,CML288和黄早四均发育正常,能够正常抽雄,节间伸长明显。15h长日照条件下CML288雄穗不能分化,节间短缩,对光周期反应敏感,黄早四抽雄略有推迟,节间伸长正常。说明CML288对光周期的敏感性明显强于黄早四,是一个对光周期敏感的热带玉米材料。通过CML288长、短日照处理相互挪移试验发现,从短日条件挪移到长日条件的植株,6叶期未见雄穗分化,7叶期、8叶期雄穗发育基本正常,9叶期雄穗发育正常,说明6～7叶期是光周期反应最敏感的时期,7～9叶期是光周期敏感的持续期,但敏感效应明显减弱。2、利用同源克隆和RACE技术,成功地分离克隆了ZmHd1的CDS序列、基因组DNA序列。CDS序列全长1310bp,具有1197bp完整的ORF阅读框,编码一条397个氨基酸残基的肽链,含有CO/Hd1家族基因2个保守的BBOX与CCT结构域,ZmHd1基因是由一个内含子与2个外显子构成,具有完整的编码区域和帽子结构、启动子和转录终止子等转录元件。玉米ZmHd1与水稻Hd1基因具有74%的序列同一性,编码的氨基酸序列具有75%的同源性,明显高于水稻与拟南芥的同源性。因此认为,玉米ZmHd1是水稻的光周期敏感基因Hd1的一个同源基因,可能是控制玉米光周期敏感反应的一个重要基因。3、黄早四与CML288 ZmHd1基因gDNA序列对比发现,在第二个外显子中发生一个从T向A的点突变,导致产生了一个翻译的终止密码子,而使具有CO/Hd1基因家族保守的CCT结构域蛋白翻译被提前终止,使C末端的核定位信号功能丧失。因此,ZmHd1基因蛋白编码区中一个核苷酸突变所产生的终止密码子,可能是光周期敏感性发生改变的一个重要机制,显示出在这个基因位点对光周期反应的敏感和钝感反应。根据黄早4在ORF之内这个早熟终止子序列,与CML288 ZmHd1序列中存在的这个核苷酸差异所产生的StuI的酶切位点,成功地开发了一个简单有效的CAPS标记,可用来测定2个玉米材料中光周期敏感基因的存在。4、ZmHd1基因序列与MaizeGDB数据库公布的B73测序的AC189064克隆序列同源性高达98～99%,由于玉米基因组测序已经基本结束,而在基因组测序的克隆中仅有一个拷贝同该基因具有这样高的同源性,因此推断该基因位于玉米第9染色体的9.03区段内,接近着丝点位置。通过RIL遗传作图群体的光周期敏感相关基因的QTLs定位分析,在这个区间检测到一个同光周期敏感反应有关的QTL,因此认为ZmHd1可能是这个光周期敏感主效QTL的一个侯选基因。Real-time PCR分析显示,ZmHd1基因进入光周期敏感时期具有上调表达的趋势,而且长日照条件的表达高于短日条件。5、以热带玉米光周期敏感时期的叶片与茎尖为材料,利用SSH技术构建了对长日照敏感而特异表达的基因文库,3000多个单克隆被有效地富集在正向光周期敏感差减文库中,为进一步分离克隆光周期敏感基因的研究搭建一个材料平台。对差减文库中分离的64个特异表达EST序列分析表明,16个EST序列与玉米已知编码的功能蛋白序列具有较高的同源性,可以初步推测其功能,可能的蛋白功能主要涉及光接受与信号转导、生长分化与发育转录因子、逆境反应等多个生理生化途径中的相关基因,31个序列与其它物种编码序列同源性较高,7个EST可能代表了新基因。6、以SSH文库的EST克隆为材料,利用同源克隆技术,成功分离克隆出了玉米PL5L15基因的cDNA序列。PL5L15基因cDNA全长1410bp,具有897bp完整的ORF阅读框,编码一条292个氨基酸残基的肽链,位于该肽链第89到272个氨基酸中含有一个保守的Clp-protease结构域。与ClpR2基因序列具有78%的同源性。因此,所克隆的PL5L15是玉米Clp蛋白酶家族的ClpR2-LIKE基因,初步推测该序列可能位于玉米第9条染色体上。Real-time PCR分析显示,在短日条件下该基因正常水平的表达促进了生殖分化,但在长日条件下持续过量高表达则抑制了生殖分化,推测可能与玉米光周期反应条件下的生殖分化有关。7、利用SSH文库的EST序列成功分离克隆出了玉米PL3K2基因的cDNA序列。序列全长639bp,具有453bp完整的阅读框,编码150个氨基酸残基的肽链,位于该肽链的第25到150个氨基酸中含有一个保守的Jacalin结构域,包含Jacalin功能域的蛋白属于Lectin基因家族成员。初步推测该序列可能位于玉米第10条染色体上。Real-time PCR分析显示,长日照逆境条件下该基因在茎尖中的表达显著高于短日照正常条件。该基因可能与玉米逆境抗性有关。更多还原

【Abstract】 Tropical and subtropical germplasms contain abundent hereditary variation to broaden Chinese maize germplasm and increase the breeding level.However,photoperiod sensitivity is the main restriction of its use in temperate zone.We have to study it in order to utilize tropical and subtropical germplasm better.CML288,a tropical maize photoperiodic sensitivity inbred line,and HuangZao No.4,a temperate maize inbred line were used as material in these studies.We studied the responses to photoperiod further in CML288 and cloned some photoperiod-related genes,ZmHd1,PL5L15 and PL3K2,which play the important roles during the transition from vegetative growth to reproductive growth,and test its transcription level by Real-Time PCR during different development leaf number stage and a whole day in photoperiod sensitivity stage.The ZmHd1 gene was mapped and deduced its function by comparing with the QTLs result of photoperiod sensitivity RILs.The main results were as follows. The inbred lines were grown in 2 different controlled photoperiod recycles,9hr and 15 hr light every day.Result showed that the HuangZao No.4 lines could perform productive growth and elongate the stem not only short-days conditon but also long-day condition.While CML288 could not development tassel and elongate the stem on long-day condition.CML288 was much more sensitive to photoperiod than HuangZao No.4.It shows that the tassel difference transferred from SD to LD condition in sixth leaf period was invisible but in seventh,the tassel development between seven-leaf and eight-leaf period were later than the tassel in short days consistently.The result indicated that CML288 was more sensitive to photoperiod between sixth and seventh leaf period.CDS and DNA sequence of ZmHd1 were isolated and cloned successfully using RACE technique.The full-length CDS sequence was 1310 bp,include a 1197bp complete ORF region,which encode a 397 amino acid peptides,owning two highly conserved BBOX and CCT domains.ZmHd1 genome gene was composed of one intron and two extron,had complete transcript elements,such as promoters and terminal sequence.It is 74 percent identifies,and 75 percent positives with Hd1 of Oryza sativa.It is an ortholog of Hd1 and related to photoperiod sensitivity. We found a premature stop codon in the second exon of ZmHd1 of Huangzao4 due to a single basepair replacement from T to A,which is not found in the same position in CML288.The translation of ZmHd1 protein had to stop ahead,and the conserved CCT domain in CO/Hd1 family was lost.So it may be an important mechanism of the change of photoperid sensitivity between tropical and temperate lines.We designed to develop a CAPS marker that depend on the difference of StuI restriction site caused by the premature stop codon to detect the photoperid sensitive gene in maize. We found the ZmHd1 gene sequence shared a high similarity with the sequence of AC189046 in Maize genome database（MaizeGDB）,and it was mapped in the chromosome 9.03,near centromere.We detected a QTL in chromosome 9.03,which is benefit to photoperiod sensitivity.So it considered that ZmHd1 was a candidate gene of this QTL region.We studied the expression of ZmHd1 by Real-Time PCR, the result showed that it has the uptrend in photoperid sensitive time,and expressed higher level in long day condition than in short day condition.A subtracted,cDNA library was constructed by the way of suppression substractive hybridization with the mRNA of leaf and shoot apical meristem of 7^th leaf stage,it contained more than 3000 clones related to photoperid sensitivity.We analyzed 64 clones,results showed that 16 EST sequences were high homologous with maize sequences related to transcription factors,signal transduction,development,flowering, adversity-stressed,and so on in GenBank.31 EST sequences were homologous with other plant genes.7 EST sequences may be the new genes.We cloned the PL5L15 gene after SSH cDNA library construction.The complete sequence was 1410bp,a predicated 897 bp open reading frame was detected in the cloned sequence,which encoded 292 amino acids,a conserved Clp-protease domain was found between 89^th and 272^nd amino acids.It was positive 78 percent with ClpR2. The RT-PCR results showed that PL5L15 is a ClpR2-LIKE gene,related to reproductive definition in photoperiodism.We cloned the PL3K2 gene based on the EST from SSH cDNA library.The complete sequence was 639bp,a deduced.453-bp open reading frame was detected in the cloned sequence,which encoded 292 amino acids,a conserved Jaclin domain was found among the amino acids.The RT-PCR results shows that P L3K2 is a subfamily gene of Letins in Maize,related to resistent response in photoperiodism.更多还原