【作者】 李莉；

【导师】 陈温福；

【作者基本信息】 沈阳农业大学 ， 作物学， 2013， 博士

【摘要】 水稻直播,是世界水稻生产发展的主要趋势之一。但是直播稻出苗难导致出苗迟,最终导致抽穗期推迟、成熟期延长,给栽培稻带来巨大的损失。中胚轴伸长与直播栽培出苗率显著相关,目前生产上所用的半矮秆栽培稻中许多品种中胚轴较短、伸长能力差,在浅土覆盖播种下,顶土出苗能力较好,在旱地直播或深土直播条件下,中胚轴短、出苗速度慢、出苗率下降,严重影响直播稻的秧苗素质。因此,发掘中胚轴较长、顶土能力强、出苗率高的水稻种质资源,才能克服短中胚轴带来的出苗不齐及其出苗难等问题,能够更好的培育出中胚轴伸长特性良好的品种,推动直播技术的发展。杂草稻(Weedy Rice)遗传变异类型丰富,适应性和抗逆性都较强,在水稻改良特别是抗性育种上是优良的种质资源库。本实验室研究发现,杂草稻具有较长的中胚轴,对于中胚轴伸长能力,杂草稻强于栽培稻,籼稻又强于粳稻。本研究以长中胚轴WR04-6和WR04-43,短中胚轴栽培稻秋光为供试材料,通过内源激素、生理酶学、对外源激素的响应及其遗传机制的研究,系统探讨了杂草稻中胚轴伸长的机理,从细胞形态学明确中胚轴伸长的实质。主要研究结果如下：1、利用人工气候箱,在黑暗条件下培养幼苗,采用石蜡切片法对杂草稻WR04-6、WR04-43和栽培稻秋光的中胚轴伸长进行了细胞形态学观察。研究发现,杂草稻幼苗的中胚轴只有在黑暗条件下才进行伸长生长,材料幼苗初期苗高的差异主要来自中胚轴的伸长。中胚轴伸长初始阶段,其细胞数目达到整个时期数目的80%以上,随后分裂增殖基本停止；杂草稻在纵向长度上细胞数目比秋光高100%,而细胞长度比秋光则长400%；在中胚轴伸长生长初期,细胞分裂增殖和细胞伸长共同促进中胚轴伸长生长,后期则依靠细胞纵向延伸促进中胚轴伸长生长。杂草稻中胚轴的伸长生长由细胞数量增加和细胞伸长共同起作用,细胞长度纵向延伸对中胚轴伸长生长起决定作用。中胚轴细胞中部细胞长度明显高于基部和顶部细胞,且长中胚轴材料较之于短中胚轴材料更加明显,而基部和顶部两区段细胞长度相差不大。2、对中胚轴内源激素测定表明,在整个中胚轴伸长生长阶段,WR04-6和WR04-43的内源GA、IAA和CTK含量都高于秋光,其中GA的差异最大,CTK的差异最小,但3个激素的含量变化趋势不同,随着幼苗生长,GA和CTK含量下降,而IAA含量持续稳步增长,不论培养几天,材料间激素含量的差异仍保持大约一致的差距。中胚轴伸长的内源激素研究表明,GA、IAA、CTK都可促进中胚轴的伸长。GA和IAA的作用较大,对中胚轴的伸长生长起着决定性作用,而CTK作用相对较小。3、中胚轴伸长的动力学研究表明,中胚轴长的材料α-淀粉酶和β-淀粉酶活性及其可溶性糖含量高,淀粉酶活性的提高,导致淀粉贮藏物质的降解以提供丰富的能量底物和结构碳架,同时可溶性糖含量与淀粉酶活性变化一致,α-淀粉酶和β-淀粉酶及可溶性糖为中胚轴的伸长提供强大的能量来源。4、中胚轴细胞壁的组份研究表明,细胞壁纤维素含量随着幼苗的生长而有所增加,且长中胚轴的杂草稻低于短中胚轴的秋光；中胚轴细胞壁酶学的研究表明,p-1,3-葡聚糖酶活性随着幼苗生长均有不同程度的增加,WR04-6的p-1,3-葡聚糖酶活性为供试材料中最高,WR04-43次之,秋光最低,与中胚轴的长度差异一致,p-1,3-葡聚糖酶活性的高低在很大程度上影响着中胚轴的伸长；细胞壁纤维素酶活性变化在整个中胚轴伸长生长阶段呈现小幅上升趋势,加速细胞壁纤维的水解,使细胞壁松弛,促进细胞的延伸；IAA氧化酶呈现稳步下降的趋势,下降幅度较大；POD酶活性在整个中胚轴生长阶段同IAA氧化酶变化趋势一致,表现出长中胚轴WR04-6、WR04-43极显著的低于秋光,细胞壁氧化酶活性较低加大了结合态IAA向游离态IAA转变的效率,使细胞中存在更多的游离态IAA,提高中胚轴内IAA含量。5、3种外源激素GA、IAA、CTK均能显著促进中胚轴的伸长,其中GA对WR04-6的促进作用最大,其次为WR04-43;IAA对WR04-43的促进作用最大,其次为WR04-6; CTK对3个材料的促进作用无显著性差别。从总体上看,GA对促进中胚轴伸长的作用最大,IAA的促进作用次之,CTK的作用最小。外加GA能刺激生长素的合成,使细胞壁松弛,细胞壁的伸展性加大,减少细胞的纵向延伸阻力,加快生长,促进细胞伸长；外加IAA促进中胚轴伸长生长,主要是提高细胞壁水解酶活性和降低细胞壁氧化酶活性,从而使细胞壁松弛,诱导细胞的伸长生长。6、杂草稻中胚轴细胞微管在快速伸长时解聚明显,微管更趋向于垂直于细胞伸长轴的T型；低浓度的黄草消(25nM)显著促进中胚轴长度的增加,高浓度黄草消(≥100nM)也抑制中胚轴伸长；1μM或更高浓度MTs稳定药剂紫杉醇显著抑制中胚轴伸长；随着中胚轴伸长,细胞微管骨架结合蛋白基因MAP65家族表达量总体趋势为下降,同时微管结合蛋白稳定基因MORl表达量上升；微管结合蛋白解聚基因KataninP60(1)、 KataninP60(2)、KataninP80表达量下降。在杂草稻中胚轴伸长过程中,动态不稳定性的MTs起着是至关重要的作用。7、以02428、WR04-6杂交后得到的F2群体,构建的水稻分子遗传图谱共包含131对标记座位,总图距约为1384.4cM,覆盖整个基因组的90.5%,标记的平均距离为10.6cM。共检测到4个控制中胚轴伸长的基因,分别位于第1、第2、第3和第4号染色体上,LOD值在2.54-3.77范围内,单个QTL对表型贡献率在6.39-16.75%之间。更多还原

【Abstract】 Rice direct sowing is one of the main trends of rice production and development in the world. The difficulty of seedling emergence of direct-seeded rice lead to delay the heading time and prolong the ripening stage which also effect full heading and yield. Significant correlation was found between the mesocotyl elongation and the emergence rate of direct sowing. The variety of semi-dwarf cultivated rice have a better emergence capacity with the shallow soil covered because of its shorter mesocotyl and poor elongation capacity. But it influenced the quality of rice shoot seriously at a dry land direct sowing or deep soil direct sowing. Therefore, excavation of the rice germplasm of longer mesocotyl and high emergence rate should be done to overcome the difficult of seeding emergence and then it is to be better breed the variety have longer mesocotyl and promote development of direct seeding.Weedy rice is fine in rice improvement especially in resistance breeding which has preferably genetic variation、adaptability and stress-resistance. Mesocotyl elongation is important for emerging, mesocotyl length materials of WR04-6and WR04-43and mesocotyl short materials of Akihikari of weedy rice were used in this study to discuss the essence of mesocotyl elongation and discuss its regulation of hormone、mechanism of physiology enzyme^response of exogenous hormone and the molecular base. The main findings are as follows:1. To observed the cytomorphology of mesocotyl in the dark condition by artificial climate chamber, the study used varieties as WR04-6、WR04-43and Akihikari,then the result showed that the elongation growth of seedling mesocotyl could occurred only at dark condition; The difference of height between varieties at seedling stage were caused by mesocotyl elongation. To observed mesocotyl cell morphology by the paraffin method and the result showed that the number of mesocotyl cell account for80%of total at whole period and then cell division and proliferation stop. The cell number of longitudinal and cell length of long mesocotyl material of weedy rice were higher100%to Akihikari, and400%than those of short mesocotyl material respectively. The cell division and elongation promoted elongation growth of seedling mesocotyl together during early stage of that and then it depended cell longitudinal extending later. The added cell number and cell elongation promoted mesocotyl elongation together while cell longitudinal extending made a decision effect on elongation growth of mesocotyl.2. Study on endogenous hormone of mesocotyl elongation show that GA、IAA and CTK could promote elongation of mesocotyl. On all stage, GA、IAA and CTK of WR04-6and WR04-43were higher than those of Akihikari. Among these endohormone, GA has the greatest difference than the IAA and CTK. Content trend of these hormonal were different. With seedling growth, GA and CTK were decline but IAA was growing continually. No matter days, differences of hormone content with materials were keep accordance and also the mesocotyl elongation.3. The dynamics research of mesocotyl elongation showed that the materials with long mesocotyl could improve activity of α-amylase and (3-amylase, and had a high content of soluble sugar. The improvement of amylase activity could result in the degradation for starch storage material, as well as priovide abundant energy substrate and carbon frame. Meanwhile, the content of solube sugar had consistent change with amylase activity.a-amylase,(3-amylase and soluble sugar provided powerful energy sources for elongation of mesocotyl.4. Research on the components of cell wall of mesocotyl showed that the content of cellulose had a increase with the seedling growth, and which of weedy rice with long mysocotyl was lower than that of akimitsu with short mesocotyl; Rusults of cell wall enzymology of mesocotyl showed that activity of β-1,3-glucanase had varying increase with seedling growth, WR04-6was the highest, WR04-43took a second place, and akimitsu was the lowest in tested materials, the changes manifested as consistent with length of mysocotyl. The activity of β-1,3-glucanase had a vary influence on elongation of mesocotyl; The changes of activity of cell wall ecllulose presented as a modest increase in the whole growth stage of hypocotyl elongation, it could accelerate hydrolysis of cell wall fiber, make cell wall loosening, and promote cell elongation; IAA oxidase was declined steadily; POD avtivity had a consistent change with IAA oxidase in the whole growth stage of mesocotyl elongation, but the weedy rice of WR04-6and WR04-43were significantly lower than akimitsu. The lower acitivity of cell wall oxidase promoted the transformation rate of conjugated IAA to free IAA. increased more free IAA in cells, and then enhanced content of IAA in mesocotyl.5. Three exogenous hormones such as GA、IAA、CTK can significantly promote mesocotyl of extension, which of GA is playing important role for promotion of WR04-6, secondly for WR04-43; IAA is playing important role for promotion of WR04-43, secondly for WR04-6; CTK was no significant difference for promotion of three materials. On the whole, GA is the most important for promoting extension of mesocotyl, IAA take a second place, effect of CTK is least. Applied GA can stimulate synthesis of IAA and make cell wall relax, extensibility of cell wall increase, and reduce longitudinal extension resistance of cell, accelerate the growth, promoting extension of cell. Applied IAA can promote extension growth of mesocotyl. The main effect was improve activity of cytohydrolist and decrease oxidase activity of cell wall, so as to make cell wall relax, induced elongation growth of cell.6. The depolymerization of mesocotyl cells of weedy rice in rapid elongation in microtube was significant, and microtubules tend t-type perpendicular to the axis of cell elongation. The application of low concentration of oryzalin (25nM) further increases the mesocotyl length in both weedy rice and cultivared rice Akihikari. In contrast, MTs-stabilizing drug taxol significantly inhibit the elongation of mesocotyl at the concentration of1μM or over. Here, we found that several genes encoding MAP65s were downregulated during the later stage of weedy rice mesocotyl elongation which may be closed relationship with the reorganization of MTs in weedy rice mesocotyl elongation. At the same time, morl, a gene coding for microtubule binding protein has higher gene expression. However, p60(1), p60(2) and p80.Which were encoding MT-serving enzymes have significant lower gene expression. In this study, we provided the evidence that dynamic instability of MTs in mesocotyl cells is essential for rice mesocotyl elongation.7. Using an F2population derived from the parents of short-mesocotyl WCVS02428and long-mesocotyl WR04-6and131SSR molecular markers built genetic linkage map.There were131pair primer had polymorphism between parents in all of935pairs SSR primer and its frequency was13.7%.The total distance was1384.4cM of the131markers and cover90.5%of whole genome, mean distance of makers was10.6cM. The four genes control the length of mesocotyl was mapped on chromosome1,2,3and4, the range of LOD was 2.54-3.77, phenotype contribution rate of single QTL was6.39-16.75%. This genetic linkage map established foundation for researching various traits of WR04-6and mesocotyl QTL更多还原