【作者】 谭彩霞；

【导师】 封超年；

【作者基本信息】 扬州大学 ， 农产品安全与环境， 2009， 博士

【摘要】 本试验以生产上推广应用的不同专用类型非糯小麦品种和缺失Wx蛋白的糯性小麦品种作为研究材料。主要研究:(1)不同专用类型小麦籽粒腺苷二磷酸葡萄糖焦磷酸化酶基因(AGPase1)、束缚态淀粉合成酶基因(GBSSI)、可溶性淀粉合成酶基因(SSSIII)、淀粉分支酶基因(SBEI)表达与淀粉合成的关系;(2)缺失不同Wx蛋白小麦籽粒中GBSSI基因表达与直链淀粉合成的关系;(3)弱筋小麦籽粒淀粉合成酶基因表达对氮素和花后温度的响应。探索不同专用类型小麦籽粒淀粉合成酶基因表达对淀粉合成与品质调控的生理机制;为小麦的高产、优质、高效栽培提供理论依据和技术参考。试验主要结果如下:1不同小麦品种籽粒淀粉合成酶基因表达、淀粉合成酶活性以及淀粉积累特点不同专用类型小麦籽粒中淀粉合成酶基因(AGPase1、GBSSI、SSSIII、SBEI)表达变化动态一致,均呈单峰曲线。于花后5天左右开始表达,15天达最大值,之后急剧下降,25天相对表达量下降至1.0左右,并相对稳定至成熟期;籽粒腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)、束缚态淀粉合成酶(GBSS)、可溶性淀粉合成酶(SSSIII)、淀粉分支酶(SBE)活性变化均呈单峰曲线,AGPase、SSS、SBE活性峰值时间出现在花后25天,GBSS活性峰值时间出现在花后30天;籽粒中淀粉及其组分含量和积累量均呈“S”型曲线变化,随着灌浆进程的推进,呈上升趋势。不同专用类型小麦间,淀粉合成酶基因(AGPase1、GBSSI、SSSIII、SBEI)相对表达量、淀粉合成酶(AGPase、GBSS、SSS、SBE)活性、淀粉积累量和积累速率大小均表现为强筋小麦中优9507>中筋小麦淮麦18>弱筋小麦宁麦9号>糯小麦Wx11,其中糯小麦Wx11籽粒中GBSSI相对表达量和GBSS活性几乎为零,籽粒中直链淀粉含量<2%;同一专用类型不同小麦品种之间,上述测定值均表现为:强筋小麦中优9507>秦麦11;中筋小麦扬麦16>淮麦18;弱筋小麦扬麦15>宁麦9号。2不同小麦品种籽粒蔗糖合成酶(SS)活性、蔗糖和可溶性总糖含量变化不同专用类型小麦籽粒中蔗糖合成酶(SS)均呈单峰曲线变化,花后20天SS活性达最大值,之后急剧下降。籽粒蔗糖和可溶性总糖含量随着灌浆进程的推进呈下降趋势。不同专用类型小麦之间籽粒蔗糖合成酶(SS)活性表现为强筋小麦中优9507>中筋小麦淮麦18>弱筋小麦宁麦9号>糯小麦Wx11,籽粒可溶性总糖和蔗糖含量表现为糯小麦Wx11>弱筋小麦宁麦9号>中筋小麦淮麦18>强筋小麦中优9507。此结果表明,强筋小麦籽粒利用同化物的能力最高,使得淀粉合成的底物最多,籽粒中淀粉积累量最高,蔗糖含量最低;糯小麦籽粒中蔗糖降解代谢能力最低,供给淀粉合成的底物最少,导致籽粒中淀粉积累量最低,蔗糖含量最高。同一专用类型不同小麦品种之间籽粒蔗糖合成酶(SS)、可溶性总糖和蔗糖含量均表现为:强筋小麦中优9507>秦麦11;中筋小麦扬麦16>淮麦18;弱筋小麦扬麦15>宁麦9号。3小麦籽粒淀粉合成酶基因表达和淀粉合成酶活性与淀粉积累的关系AGPase1和SSSIII基因相对表达量最大值与AGPase、GBSS、SSS、SBE、SS酶活性峰值的相关性均达显著或极显著水平;GBSSI基因相对表达量最大值与AGPase和SS酶活性峰值的相关性达显著水平,与GBSS、SSS、SBE酶活性峰值的相关性未达显著水平; SBEI基因相对表达量最大值与SSS和SBE酶活性峰值相关性达极显著水平,与AGPase、GBSS、SS酶活性峰值的相关性未达显著水平;另外AGPase1、GBSSI、SSSIII、SBEI基因相对表达量和AGPase、GBSS、SSS、SBE酶活性与籽粒直、支链淀粉及总淀粉积累速率均呈极显著正相关。说明这四种淀粉合成酶基因和淀粉合成酶共同对籽粒淀粉的合成起作用;AGPase1、SSSIII、SBEI基因可能主要通过转录水平控制籽粒淀粉的合成,而GBSSI基因可能主要通过转录后水平控制籽粒直链淀粉的合成。4缺失不同Wx蛋白小麦籽粒中GBSSI基因表达与直链淀粉合成的关系缺失不同Wx蛋白小麦籽粒中GBSSI基因相对表达量、GBSS活性、直链淀粉积累量均表现为:正常型>缺A型>缺D型>缺B型>缺AB型>缺ABD型;籽粒淀粉最终粘度、反弹值、糊化温度在缺失不同Wx蛋白小麦品种之间表现顺序与其一致;淀粉峰值粘度、低谷粘度、稀懈值、沉降值表现顺序与其相反。相关分析表明,淀粉膨胀势与直链淀粉含量、反弹值和糊化温度呈显著或极显著负相关,与淀粉峰值粘度、低谷粘度呈显著或极显著正相关。5弱筋小麦籽粒淀粉合成酶基因表达对氮素的响应弱筋小麦15籽粒中淀粉合成酶基因(AGPase1、GBSSI、SSSIII、SBEI)相对表达量、淀粉合成酶(AGPase、GBSS、SSS、SBE)活性、淀粉积累量以及部分淀粉粘度参数值在相同的氮肥运筹比例时,施氮量为150kg hm-2～210kg hm-2范围时,随着施氮量的增加,均呈上升趋势;当施氮量超过210kg hm-2时,随着施氮量的增加,上述指标值降低;相同的施氮量水平下,当追施氮肥适当前移时,弱筋小麦扬麦15籽粒中上述指标值增加。6弱筋小麦籽粒淀粉合成酶基因表达对花后温度的响应花后不同时期经25℃和35℃处理后,弱筋小麦扬麦15籽粒中淀粉合成酶基因相对表达量(AGPase1、GBSSI、SSSIII、SBEI)、淀粉合成酶活性(AGPase、GBSS、SSS、SBE)、淀粉积累量以及部分淀粉粘度参数值均下降,其下降幅度在各处理间的顺序表现为:花后5-7d>花后10-12d>花后15-17d>花后20-22d>花后25-27d>花后30-32d>花后35-37d,花后5-7d高温处理下降幅度最大。同一时期经不同温度处理后,籽粒中上述指标值下降幅度均表现为:35℃>25℃,35℃处理下降幅度最大,25℃处理与CK差异不显著。花后5-7d经35℃处理后,籽粒中除GBSSI和GBSS外,其它3种淀粉合成酶基因相对表达量和相应酶活性到达峰值的时间均前移。GBSSI和GBSS对温度最为钝感,SSSIII基因和SSS对温度最为敏感。7荧光定量PCR技术方法的改进荧光定量PCR是一种在PCR反应体系中加入荧光基团,利用荧光信号实时监测整个PCR进程,最后通过标准曲线对未知模板进行定量分析的方法。该方法的优点在于它能监测任何dsDNA序列的扩增,不需要探针的设计,使检测方法变得简便,同时也降低了检测的成本。本试验利用DNA消化酶去除mRNA中的DNA,以防止DNA污染;根据荧光定量PCR的要求严格设计引物,避免产生引物二聚体;把不同模板的cDNA混合进行稀释制作标准曲线;优化荧光定量PCR的反应体系,采用两步法RT-PCR进行荧光定量检测。这种制作方法可以准确反映反应体系中反转录和扩增过程的效率,使结果更为可信。更多还原

【Abstract】 The expression levels of ADP-Glc Pyrophosphorylase gene(AGPase1), Granule-Bound Starch Synthase gene(GBSSI), Soluble Starch Synthase gene(SSSIII), and Starch Branching Enzyme gene(SBEI) and their relationships with the starch synthesis in wheat grains of different gluten types were investigated with non-waxy wheat cultivars and waxy wheat varieties. The relationship between GBSSI gene expression and amylose synthesis in wheat grains with different missing Wx proteins, and the responses of the expression levels of starch synthase genes in weak-gluten wheat grains to nitrogen and high temperature after anthesis were analyzed . These studies are of crucial importance both in exploring the physiological mechanism for starch synthase gene expression regulating starch synthesis and starch quality in wheat grains with different gluten types and in providing theoretical basis and technical guidance for efficient production in wheat grains with high yield and goodquality. The main results were as follows.1. Changes of starch synthase genes expression levels, starch synthases activities and starch accumulation in grains of different wheat cultivars during grain fillingSingle-peak trends were observed in the expression levels of AGPase1, GBSSI, SSSIII and SBEI during grain filling. The expressions of these genes were first detected on DAA 5 (the 5th day after anthesis), reached the maximum on DAA 15, and declined significantly from DAA 15. The lowest expression levels of starch synthase genes were recorded during the period of DAA 30-40, only at around 1.0. The activities of AGPase, SSS, GBSS, and SBE performed in the pattern of a single-peak curve during grain filling. AGPase, SSS and SBE activities reached their peaks on DAA 25 and GBSS activity reached its maximum on DAA 30. The starch content and accumulation in grains rose gradually after anthesis,Among the different gluten types of wheat varieties, the expression levels of starch synthase genes, the activities of starch synthase, and starch accumulation were recorded in a descending order of Zhongyou9507 (a strong-gluten cultivar), Huaimai18 (a medium-gluten cultivar), Ningmai9 (a weak-gluten cultivar), and Wx11 (a Waxy variety). The expression of GBSSI and the activity of GBSS in Wx 11 grains were maintained at very low levels approaching zero, which led to amy-lose content in Wx11 grains were lower than 2%. Between different wheat varieties with the same gluten type, the order of above-mentioned parameters was: Zhongyou9507 > Qinmai11 in strong-gluten cultivars; Yangmai16 > Huaimai18 in medium-gluten cultivars; Yangmai15 > Ningmai9 in weak-gluten cultivars.2 Changes of SS activity, sucrose and total soluble sugar contents in grains of different wheat cultivars during grain fillingThe activities of SS performed in the pattern of a single-peak curve in wheat grains with different gluten types during grain filling. Its peak appeared on DAA 20. The sucrose and total soluble sugar contents in wheat grains dropped constantly respectively after anthesis and at early grain filling stage.Among different gluten types of wheat varieties, the activity of SS in a descending order of Zhongyou9507, Huaimai18, Ningmai9, and Wx11, and the order of sucrose and total soluble sugar contents in wheat grains were expressed as Wx11 > Ningmai9 > Huaimai18 > Zhongyou9507. The results showed that a large amount of sugar in strong-gluten wheat grains had been converted into the substrate for starch synthesis, therefore the starch accumulation in its grains was much higher than that in the grains of the other three gluten types. In waxy wheat grains there were a large amount of sugar which was incapable of being converted into the substrate for starch synthesis. Hence, the starch accumulation in its grains was much lower than that in non-waxy wheat grains. Between different wheat varieties with the same gluten type, the above mentioned parameters were in the following order: Zhongyou9507 > Qinmai11 in strong-gluten cultivars; Yangmai16 > Huaimai18 in medium-gluten cultivars; Yangmai15 > Ningmai9 in weak-gluten cultivars.3. Relationship of the expression levels of starch synthase genes with the activities of starch synthases and starch accumulation in wheat grainsThe relationship between the maximum expression levels of AGPase1, SSSIII and the peak activities of AGPase, GBSS, SSS, SBE, SS was significantly correlated at 0.01 or 0.05 levels. The maximum expression level of GBSSI was significantly correlated with the peak activities of AGPase and SS, while there were no significant correlations between it and the peak activities of GBSS, SSS, and SBE. The maximum expression level of SBEI was highly significantly correlated with the peak activities of SSS and SBE, (p<0.01), and insignificantly correlated with the peak activities of AGPase, GBSS, and SS. The expression levels of AGPase1, GBSSI, SSSIII, and SBEI and the activities of AGPase, GBSS, SSS, and SBE were highly significantly correlated with the accumulatiing rates of amylase, amylopectin, and total starch. These suggested that starch synthase genes and starch synthases played collaborative roles in starch synthesis in wheat grains. AGPase, SSS,and SBE might probably control starch synthesis at transcriptional level, and GBSSI might probably control starch synthesis at post-transcriptional level.4 Relationship between GBSSI gene expression and amylose synthesis in wheat grains with different missing Wx proteinsThe expression level of GBSSI, GBSS activity, and amylase content were recorded in the following order: normal type > single null allels of Wx-A1 > single null allels of Wx-D1 > single null allels of Wx-B1 > double null allels of Wx-A1 and Wx-B1 > waxy type. Similar trends were observed in final viscosity, rebound value, and gelatinization temperature, while opposite trends were measured in peak viscosity, trough viscosity, breakdown, and sedimentation value.5 Responses of starch synthase gene expressions in weak-gluten wheat grains to nitrogenThe expression levels of starch synthase genes (AGPase1, GBSSI, SSSIII, SBEI), the activities of starch synthases (AGPase, GBSS, SSS, SBE), and starch accumulation rose in the grains of Yangmai15, a weak-gluten wheat cultivar, with the increase of nitrogen application amount varying from 150kg hm-2 to 210kg hm-2. When nitrogen application amount was higher than 210kg hm-2, those decreased. The expression levels of starch synthase gene, the activities of starch synthase, and starch accumulation were stimulated when topdressing N percentage decreased reasonably.6 Responses of starch synthase gene expression in weak-gluten wheat grains to high temperature after anthesisThe expression levels of starch synthase genes (AGPase1, GBSSI, SSSIII, SBEI), the activities of starch synthases (AGPase, GBSS, SSS, SBE), and starch accumulation in the grains of Yangmai15 were significantly inhibited when the wheat plants were subjected to high temperatures during grain filling. The decrements of these parameters among different high temperature treating periods were in the following order: 5-7DAA > 10-12DAA > 15-17DAA > 20-22DAA > 25-27DAA > 30-32DAA > 35-37DAA. Compared to 25℃, the inhibitive effects were more significant at 35℃. Similar trends were found in these parameters for the treatment of 25℃and the control. The expression levels of AGPase1, SSSIII, and SBEI, and the activities of AGPase, SSS, SBE reached their peaks on earlier dates when subjected to 35℃on 5-7 DAA. GBSSI and GBSS were insensitive to high temperatures. SSSIII and SSS were sensitive to high temperatures.7 Improvement of real-time quantitative PCR techniqueReal-time quantitative PCR (RT-PCR) is a kind of nucleicacid technology, which can monitor the entire real-time PCR process through the accumulation of fluorescence signal and analysis the unknown sample through the standard curve. Therefore, it has the characteristics of real-time monitoring, simple and cost saving. In this experiment, a series of measures had been taken to further improve RT-PCR technology, which included removing DNA in mRNA with DNA enzyme, strictly designing primers in accordance with the requirements of RT-PCR, mixing different template cDNA to make standard curve, optimizing the reaction system of RT-PCR and the use of two-step RT-PCR reaction. These measures above mentioned can accurately reflect the process of reverse transcription and amplification.更多还原