【作者】 肖辉海；

【导师】 陈良碧；

【作者基本信息】 湖南农业大学 ， 植物学， 2005， 博士

【摘要】 隐性长穗颈(elongated-uppermost-internode，简称eui)性状是解决不育系包颈现象的重要要遗传资源。有关温敏雄性核不育水稻中eui基因的遗传规律等方面已有深入研究，但环境因素对eui基因表达的影响、穗颈节间伸长的细胞学机制、eui基因的特异性cDNA片段的克隆等方面的研究还未见报道。本文以培矮64S为对照，研究温度对隐性长穗颈温敏雄性核不育水稻eui基因表达的影响，eui基因促进穗颈节间伸长的细胞学机制，以及利用SSH技术分离隐性长穗颈温敏雄性核不育水稻长选3S穗颈节间特异表达的cDNA片段。主要研究结果如下：(1)自然条件下长选3S茎秆高度比培矮64S的高24.3cm，主要是由倒一节间(即穗颈节间)的伸长所致，长选3S的倒一节间比培矮64S的长16.6cm，增加的长度占长选3S倒一节间长度的47.3％。倒二节间和倒三节间也有一定的伸长，倒四节间与对照基本一致。穗颈伸出剑叶鞘的长度由倒一节间长度和剑叶鞘长度决定，长选3S剑叶叶鞘与培矮64S相比增长2.8cm，表明隐性长穗颈基因对叶鞘伸长有一定的促进作用，但这种促进作用远小于对穗颈节间伸长的促进作用，使隐性长穗颈不育系长选3S的包颈程度比培矮64S的大大降低或不包颈。(2)长选3S穗颈伸出叶鞘的长度与始花当天的日均温度无关，而与始花前20-9 d自然条件下的日均温度呈负相关，其中以始花前17-12d(花粉母细胞形成至减数分裂期)日均温度负相关性最显著。始花前不同时段24℃处理的结果表明：与对照相比，包穗现象有不同程度的减轻，有5个处理使穗全部伸出剑叶叶鞘，其中以始花前17-12 d(即花粉母细胞形成至减数分裂期)温度处理伸颈伸出长度最大，人工温度处理的结果与自然条件下温度与穗颈伸出度的相关性分析一致，因此，始花前17-12 d(即花粉母细胞形成至减数分裂期)是隐性长穗颈eui基因表达对温度最敏感的时期。在eui基因表达对温度最敏感的时期进行28℃、26℃、24℃、22℃四种人工温度处理，28℃条件下eui基因表达受抑制，有30％的颖花被包在剑叶叶鞘中；26℃-22℃条件下eui基因表达，且穗颈伸出剑叶鞘的长度随温度的降低而增加。(3)在幼穗分化的第Ⅵ期，长选3S和培矮64S之间穗颈节间长度相差不大，从第Ⅶ期开始，长选3S穗颈节间伸长的速度比培矮64S的快。而从第Ⅷ期至始花当天，长选3S穗颈节间伸长的速度显著快于培矮64S的，这一时段长选3S穗颈节间日均伸长的速度是培矮64S的2.2倍，穗颈节间伸长的长度是培矮64S的2.5倍。始花后两者穗颈节间伸长速度逐渐减慢，最后停止伸长。不同发育时期穗颈节间的细胞平均长度和细胞个数也呈现相似的变化规律，即长选3S穗颈节间薄壁细胞个数和长度在Ⅵ期时与培矮64S并无很大差异，随着幼穗的不断发育，细胞长度和细胞个数的差异越来越大，到定长时差异达到最大。长选3S穗颈节间薄壁细胞个数的增加从Ⅶ期到Ⅷ期至始花当天是呈直线式上升趋势，而到了始花之后细胞个数增加速度减慢；薄壁细胞的长度在Ⅶ期至始花当天也显著的增加，但从始花到定长这一阶段细胞长度呈缓慢增长的趋势。人工温度(24℃)条件下长选3S穗颈节间伸长的动态变化、穗颈节间的细胞平均长度及细胞个数的变化规律等与自然条件下所得的结果基本一致。综上所述，长选3S穗颈节间的伸长主要是由幼穗分化前期细胞分裂和细胞伸长共同作用的结果。(4)自然条件下长选3S定长穗颈节间的长度比对照培矮64S的长18.0 cm，其纵列外层和内层薄壁细胞个数分别比培矮64S的多1 248个和580个；外层和内层薄壁细胞的平均长度分别比培矮64S的长23.3μm和38.3μm，尤其是中部区段细胞的平均长度差值最大，长选3S穗颈节间中部区段外层和内层薄壁细胞的平均长度分别比对照的长24.8μm和48.7μm。说明隐性长穗颈温敏核不育水稻穗颈节间伸长是细胞数目增多和细胞长度增加双重作用所致，其后者作用更显著。在不同温度处理后，长选3S穗颈节间最内和最外层薄壁细胞个数和细胞平均长度随处理温度升高逐渐减少；与22℃处理的材料比较，28℃处理的材料穗颈节间短11.79 cm，其纵列最外和最内层薄壁细胞数目分别少771个和292个，细胞平均长度分别短13.9μm和24.6μm。表明eui基因促进细胞分裂和细胞伸长的作用随处理温度的升高逐渐减弱。(5)以培矮64S为对照群体，长选3S为目标群体，进行抑制差减杂交。用经过PA cDNA差减的CX cDNA构建一个差减文库。该文库包含约130个独立的重组克隆，插入片段平均大小为185 bp左右。(6)采用差减前的培矮64S cDNA和长选3S cDNA以及正向／反向差减杂交后的cDNA为模板标记探针，对随机挑取的96个重组克隆进行差示筛选，结果筛选出与差减前的长选3ScDNA探针和正向差减的cDNA探针都有杂交信号的特异表达克隆4个，只与正向差减的cDNA探针有杂交信号的特异表达克隆16个，共计20个，从20个候选阳性克隆中随机挑取8个进行Northern杂交，证实其中1个候选阳性克隆为长选3S穗颈节间特异表达的cDNA片段。但该基因是否为水稻中的高秆隐性基因(即eui基因)以及它在隐性长穗颈不育水稻穗颈节间伸长过程中的作用，还有待于在获得该cDNA片段的全长、进行更有效地同源性比较和对其进行功能研究后才能确定。综上所述，eui基因具有显著地促进穗颈节间内层薄壁细胞伸长的作用；该基因的表达与育性敏感期(即花粉母细胞形期至减数分裂期)的日均温度呈极显著的负相关，在此时期内如果日均温度在24℃-26℃之间，则能同时满足不育基因和eui基因的充分表达。因此，隐性长穗颈不育水稻在大田生产应用时要注意选择适宜的时间段和地域，既保证隐性长穗颈eui基因能够充分表达，又确保其育性不发生波动；在隐性长穗颈eui基因感温的敏感时期若遇到持续高温天气，在抽穗时则可通过适当喷施赤霉素以确保穗颈节伸出。有关eui基因全序列的获得和其功能确定还需要作大量的研究工作。更多还原

【Abstract】 The poor exserted panicle of the male sterile line in rice is one ofmain problems in hybrid seed production. In order to overcome the panicleenclosed, spraying GA₃ is the only sure to make panicle exserted. However,elongated uppermost internode（eui） gene is very useful for seedproduction of hybrid rice. Changxuan 3S was thermo-sensitive malesterile（TGMS） rice with eui gene selected from seeds of Peiai 64Sirradiated by 350 Gy⁶⁰Coγ-ray. To provide science evidence and theorysupport for exploiting and utilizing TGMS rice Changxuan 3S with eui gene,the correlativity between panicle exsertion of Changxuan 3S and naturetemperature was analyzed, and effect of temperature on eui gene expressionwas studied, and the cell number and length were researched. At the sametime, specifc expression cDNA seqence of the uppermost internode forchangxuan 3S were isolated and cloned by using supperssion subtractivehybridization （SSH）. The main results were showed as below:The culm length of Changxuan 3S was 24.3 cm longer than that of Peiai64S, the increased culm length for Changxuan 3S was mainly due to theelongation of the uppermost internode. The length of the uppermostinternode for Changxuan 3S was 16.6cm longer than that of Peiai 64S, andthe panicle exsertion was 13.8cm longer than that of Peiai 64S at naturaltemperature condition. Cytological studies on Changxuan 3S and Peiai 64Sshowed that the number of external and internal parenchyma cells in theuppermost internode of Changxuan 3S were 1 248 and 580 more than thoseof Peiai 64S respectively and the length 23.3μm and 38.3μm longer respectively. The number and length of cell had no obvious difference inbasal and top sections, but the length of parenchyma cell in middle sectionof the uppermost internode in Changxuan 3S was much longer than that inPeiai 64S, i.e. the two kinds of parenchyma cells were 24.8μm and 48.7μm longer respectively. Those resulted in 16.6 cm longer for uppermostinternode and 13.8 cm longer for panicle exserted in Changxuan 3S thanthat in Peiai 64S. It is, therefore, evident that elongation of uppermostinternode of Changxuan 3S is due to the increase of cell number and cellelongation. And the elongation of later was more significant.The length of panicle exsertion of Changxuan 3S was negativelycorrelated with a daily average temperature during 20-9d beforeflowering and with a most effective period between 17-12d at naturaltemperature condition.The optimal panicle exsertion of Changxuan 3S was recorded in thisstage of 17-12d before flowering when treated for 6 d at 24℃. The resultof artificial temperature treatment was the same as the correlationanalysis between panicle exsertion and daily mean temperature of 17-12dbefore flowering at natural temperature condition, i.e. the criticalperiod of eui gene expression to temperature was the period of preblooming17-12d （forming pollen mother cell and meiosis）Changes in length of elongated uppermost internodes under thecondition of constant 24℃showed that the internodes began elongation atpreblooming 12d. At preblooming 8d, high elongation of the internodes wasrecorded, and the elongation rate of internodes for Changxuan 3S wasfaster than that of Peiai 64S. The fastest period of internode elongationof Changxuan 3S was the fourth day preblooming to 0 day （flowering） withthe rate of 5.8 cm per day, which contributed 63.38％of the total length,while Peiai 64S had 2.65 cm elongation per day and 50.72％of the totallength. The internodes elongation rate decreased obviously afterflowering, and stopped elongation at the third day after flowering. The results demonstrated that the difference of internodes elongation betweenChangxuan 3S and Peiai 64S was mainly the fourth day preblooming toflowering and the mean elongation rate of internodes for Changxua3S was2.2 times faster than that of Peiai 64S.When the temperature treatments at 22℃, 24℃, 26℃or 28℃wereimposed on Changxuan 3S during the most sensitive period of eui geneexpression, the expression of eui gene was depressed under the temperatureof 28℃, whereas improved at the temperature of 22℃, 24℃, 26℃, in thatthe lower the temperature, the longer the panicle exsertion. Afterdifferent temperature treatments, the cell number and mean length ofelongated uppermost internodes were lower along with increasingtemperatures in Changxuan 3S. After treated at 28℃, the number ofoutermost and innermost parenchyma cells was 771 and 292 respectively morethan those of 22℃. The mean length of cells was 13.9μm and 24.6μmrespectively shorter than those of 22℃. The mean length of the two cellshad no obvious difference in basal and top sections, but the mean lengthof outermost and innermost parenchyma cells was 18.0μm and 35.4μmrespectively shorter than those of 22℃in middle sectionsA subtracted cDNA library specific to Changxuan 3S was constructedby SSH and pGEM-T Easy Vector cloning, which consisted of 130 individualrecombinant clones. Each clone had an insert with the average size of 185bp, and the frequency of inserting was 100％.Using PCR-select differential screening kit, the 96 recombinantclones of the constructed above cDNA library were chosen randomly and thenhybridized with forward and reverse subtracted probes and unsubtractedprobes. The results showed that 20 positive clones specific to theelongated uppermost iternode of Changxuan 3S were obtained. To confirmtheir positive results, 8 clones of them were chosen randomly from theabove positive clones and analyzed by Northern blot hybridization. Theresults showed that the probe built from 1 candidate clone hybridized only with Changxuan 3S mRNA, 4 clones hybridized with Changxuan 3S and Peiai64S mRNA, while the other 3 clones had no hybridization signals withChangxuan 3S and Peiai 64S mRNA, which probably represented cDNA clonesexpressed by low-abundance transcripts in Changxuan 3S.The positive clone specific to the elongated uppermost iternode ofChangxuan 3S was sequenced. The relationship between the gene expressionand elongation of the uppermost internode for Changxuan 3S was discussed.更多还原