不同品种青稞籽粒灌浆期淀粉代谢酶活性及基因表达模式的研究
发布时间：2018-09-13 06:28
【摘要】：本实验选用糯性青稞品种甘垦5号和非糯性青稞品种北青6号和昆仑12号作为试验材料,以双波长分光光度法测定3个青稞品种籽粒灌浆期中直链淀粉与支链淀粉含量的动态变化;通过测定酶活分析研究了淀粉代谢的关键酶活性变化;通过RT-PCR研究并分析了淀粉代谢关键酶编码基因的表达量,以及不同基因表达量之间和基因表达量与对应酶活的相互关系。结果表明:1.随着籽粒灌浆期的进行甘垦5号、北青6号和昆仑12号的支链淀粉的含量、总淀粉的含量随籽粒灌浆期进行而逐渐增加,北青6号和昆仑12号的直链淀粉的含量也随之逐渐增加。其中北青6号和昆仑12号的支链淀粉前期积累速率较快,而直链淀粉后期积累速率较快,最终导致支链淀粉和直链淀粉的比例逐渐下降,花后15天左右,其比例已无差异。2.甘垦5号、北青6号和昆仑12号籽粒灌浆过程中催化淀粉合成的关键酶:淀粉合成酶AGPP、SSS、GBSS和SBE的活性变化均呈单峰曲线,北青6号的AGPP和昆仑12号的GBSS的活性在花后25天左右达到峰值,而甘垦5号的AGPP、SSS和SBE的活性和北青6号的SSS、GBSS和SBE的活性以及昆仑12号的AGPP、SSS、和SBE均在花后20天左右达到峰值;而在籽粒灌浆过程中α-淀粉酶则出现升-降-升-降的变化趋势,即在花后10天左右出现第一个峰值,花后20天左右降到最低,花后25天左右,再次出现峰值,之后开始下降,而β-淀粉酶基本上是呈先下降后上升的趋势,在花后20天左右活性值最低。3.相关性分析结果表明,北青6号籽粒灌浆期淀粉合成酶AGPP、SSS、GBSS和SBE的活性变化与直链淀粉含量的变化呈显著正相关,北青6号和昆仑12号籽粒灌浆直链淀粉、支链淀粉和总淀粉积累速率与AGPP、GBSS、SSS和SBE活性变化显著或极显著正相关。在甘垦5号中未检测到直链淀粉的积累,甘垦5号籽粒灌浆期的支链淀粉和总淀粉积累速率与AGPP和SSS呈显著或者极显著正相关。而北青6号和昆仑12号籽粒灌浆期的直链淀粉、支链淀粉和总淀粉的积累速率与β-淀粉酶活性呈显著或极显著负相关。甘垦5号籽粒灌浆期的支链淀粉和总淀粉积累速率与α-淀粉酶呈显著负相关。青稞籽粒灌浆过程中淀粉含量积累是由于淀粉合成酶活性的增强,而不是分解酶活性的减弱。因此,青稞籽粒中淀粉积累是淀粉代谢酶(合成和分解方向)综合作用的结果。4.基因的表达量分析结果表明:甘垦5号、北青6号和昆仑12号的淀粉合成编码基因的表达模式大致可分为两种曲线模式,即低-高-低的单峰曲线和高-低-高-低的双峰曲线。其中GBSSI在糯性青稞品种甘垦5号中一直保持低量表达,但在非糯性材料北青6号和昆仑12号中呈现单峰曲线的表达模式;AGPP、GBSSI、SSSIII和SBEI在胚乳淀粉合成时期表达量较高;GBSSII后期表达量较高,但在整个灌浆过程中表达量均较低。淀粉分解编码基因中,AMY4的mRNA在3个青稞品种整个开花过程中呈现逐渐升高的趋势外,AMY1、AMY2和AMY3均呈现在开花前期表达量较高,开花中期表达量相对较低但保持稳定,开花后期表达量逐渐回升的趋势。青稞籽粒灌浆过程中BAM1和BAM2在开花前期表达量较高,而BAM6和BAM7则在开花后期有较高的表达量,BAM4和BAM5在整个灌浆过程中则为恒量表达。可通过分解酶基因表达量解释淀粉分解酶活性先降低后上升的变化趋势。
[Abstract]:In this experiment, glutinous and non-glutinous highland barley varieties Ganken 5 and Kunlun 12 were selected as experimental materials, and the dynamic changes of amylose and amylopectin contents in grain filling stage of three highland barley varieties were determined by dual-wavelength spectrophotometry. The expression of genes encoding key enzymes in starch metabolism was studied by RT-PCR, and the relationship between gene expression and corresponding enzyme activity was analyzed. The results showed that: 1. With the content of amylopectin in Ganken 5, Beiqing 6 and Kunlun 12 during grain filling, the total starch content increased with grain filling. The amylopectin content of Beiqing 6 and Kunlun 12 increased gradually, and the amylopectin accumulation rate of Beiqing 6 and Kunlun 12 was faster in the early stage, but the amylopectin accumulation rate was faster in the later stage, which eventually led to the gradual decrease of the amylopectin and amylopectin proportion, and the proportion had no difference about 15 days after anthesis. 2. The activities of the key enzymes for starch synthesis during grain filling: starch synthase AGPP, SSS, GBSS and SBE showed a single peak curve. The activities of AGPP and GBSS of Beiqing 6 and Kunlun 12 reached a peak value about 25 days after anthesis, while those of AGPP, SSS and SBE of Ganken 5 and SSS, GB of Beiqing 6 reached a peak value. The activity of SS and SBE, AGPP, SSS, and SBE of Kunlun 12 reached their peak value about 20 days after anthesis, while the change trend of alpha-amylase appeared during grain filling, that is, the first peak value appeared about 10 days after anthesis, the lowest value appeared about 20 days after anthesis, the peak value appeared again about 25 days after anthesis, and then began to decline. The activity of amylase AGPP, SSS, GBSS and SBE in grain filling stage of Beiqing 6 was positively correlated with amylose content. The amylose content in grain filling stage of Beiqing 6 and Kunlun 12 was positively correlated with amylose content. Amylose and total starch accumulation rates were significantly or extremely significantly positively correlated with AGPP, GBSS, SSS and SBE activities. Amylose accumulation was not detected in Ganken 5. Amylose and total starch accumulation rates at grain filling stage of Ganken 5 were significantly or very significantly positively correlated with AGPP and SSS. The accumulation rates of amylopectin, amylopectin and total starch were significantly or extremely significantly negatively correlated with the activity of beta-amylase. The accumulation rates of amylopectin and total starch were significantly negatively correlated with the activity of alpha-amylase at grain filling stage of Ganken 5. The accumulation of starch content during grain filling was due to the increase of amylase synthase activity, not to the increase of amylase synthase activity. Therefore, starch accumulation in highland barley grains is the result of the synthesis and decomposition of starch metabolizing enzymes. 4. The results of gene expression analysis show that the expression patterns of starch synthesis coding genes in Ganken 5, Beiqing 6 and Kunlun 12 can be divided into two kinds of curve patterns, that is, low-high-low single peak curve. The expression of GBSSI in waxy highland barley variety Ganken 5 was low, but in non-waxy materials Beiqing 6 and Kunlun 12, the expression of AGPP, GBSSI, SSSIII and SBEI was high at the endosperm starch synthesis stage; the expression of GBSSII was high at the late stage, but in the whole irrigation stage. The expression of AMY4 mRNA increased gradually in the whole flowering process of three highland barley varieties. The expression of AMY1, AMY2 and AMY3 were higher in the early flowering stage, lower but stable in the middle flowering stage, and gradually increased in the late flowering stage. During grain filling, BAM1 and BAM2 were highly expressed in the early flowering stage, while BAM6 and BAM7 were highly expressed in the late flowering stage, while BAM4 and BAM5 were constant in the whole filling stage.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S512.3
[Abstract]:In this experiment, glutinous and non-glutinous highland barley varieties Ganken 5 and Kunlun 12 were selected as experimental materials, and the dynamic changes of amylose and amylopectin contents in grain filling stage of three highland barley varieties were determined by dual-wavelength spectrophotometry. The expression of genes encoding key enzymes in starch metabolism was studied by RT-PCR, and the relationship between gene expression and corresponding enzyme activity was analyzed. The results showed that: 1. With the content of amylopectin in Ganken 5, Beiqing 6 and Kunlun 12 during grain filling, the total starch content increased with grain filling. The amylopectin content of Beiqing 6 and Kunlun 12 increased gradually, and the amylopectin accumulation rate of Beiqing 6 and Kunlun 12 was faster in the early stage, but the amylopectin accumulation rate was faster in the later stage, which eventually led to the gradual decrease of the amylopectin and amylopectin proportion, and the proportion had no difference about 15 days after anthesis. 2. The activities of the key enzymes for starch synthesis during grain filling: starch synthase AGPP, SSS, GBSS and SBE showed a single peak curve. The activities of AGPP and GBSS of Beiqing 6 and Kunlun 12 reached a peak value about 25 days after anthesis, while those of AGPP, SSS and SBE of Ganken 5 and SSS, GB of Beiqing 6 reached a peak value. The activity of SS and SBE, AGPP, SSS, and SBE of Kunlun 12 reached their peak value about 20 days after anthesis, while the change trend of alpha-amylase appeared during grain filling, that is, the first peak value appeared about 10 days after anthesis, the lowest value appeared about 20 days after anthesis, the peak value appeared again about 25 days after anthesis, and then began to decline. The activity of amylase AGPP, SSS, GBSS and SBE in grain filling stage of Beiqing 6 was positively correlated with amylose content. The amylose content in grain filling stage of Beiqing 6 and Kunlun 12 was positively correlated with amylose content. Amylose and total starch accumulation rates were significantly or extremely significantly positively correlated with AGPP, GBSS, SSS and SBE activities. Amylose accumulation was not detected in Ganken 5. Amylose and total starch accumulation rates at grain filling stage of Ganken 5 were significantly or very significantly positively correlated with AGPP and SSS. The accumulation rates of amylopectin, amylopectin and total starch were significantly or extremely significantly negatively correlated with the activity of beta-amylase. The accumulation rates of amylopectin and total starch were significantly negatively correlated with the activity of alpha-amylase at grain filling stage of Ganken 5. The accumulation of starch content during grain filling was due to the increase of amylase synthase activity, not to the increase of amylase synthase activity. Therefore, starch accumulation in highland barley grains is the result of the synthesis and decomposition of starch metabolizing enzymes. 4. The results of gene expression analysis show that the expression patterns of starch synthesis coding genes in Ganken 5, Beiqing 6 and Kunlun 12 can be divided into two kinds of curve patterns, that is, low-high-low single peak curve. The expression of GBSSI in waxy highland barley variety Ganken 5 was low, but in non-waxy materials Beiqing 6 and Kunlun 12, the expression of AGPP, GBSSI, SSSIII and SBEI was high at the endosperm starch synthesis stage; the expression of GBSSII was high at the late stage, but in the whole irrigation stage. The expression of AMY4 mRNA increased gradually in the whole flowering process of three highland barley varieties. The expression of AMY1, AMY2 and AMY3 were higher in the early flowering stage, lower but stable in the middle flowering stage, and gradually increased in the late flowering stage. During grain filling, BAM1 and BAM2 were highly expressed in the early flowering stage, while BAM6 and BAM7 were highly expressed in the late flowering stage, while BAM4 and BAM5 were constant in the whole filling stage.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S512.3
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