摘要
本文选用糯性和非糯性两种不同类型小麦为材料,通过田间试验考察了它们在灌浆过程中直、支链淀粉和总淀粉的积累量、积累速率变化及参与淀粉合成的关键酶活性变化;应用葡聚糖凝胶层析的方法研究了支链淀粉不同链长的积累动态过程;应用荧光实时定量技术研究了淀粉合成关键酶编码基因在转录水平上的调节机制,并分析了它们之间的相互关系。结果表明:
1.灌浆期,糯性和非糯性小麦籽粒中的直(非糯)、支链淀粉和总淀粉的积累量均呈“S”形曲线变化;直、支链淀粉和总淀粉的积累速率均呈先增加后降低的单峰曲线变化,并且均在花后20天左右达最大积累速率;另外,直、支链淀粉的最终积累量主要取决于最大积累速率和平均积累速率的大小,与淀粉积累的起始时间关系不大。
2.糯性和非糯性小麦籽粒淀粉合成过程中催化淀粉合成的关键酶:腺苷二磷酸葡萄糖焦磷酸化酶(AGPP)、可溶性淀粉合成酶(SSS)、束缚态淀粉合成酶(GBSS)和淀粉分支酶(SBE)的活性变化均呈单峰曲线,活性峰值基本上都出现在花后20~25天左右;
3.相关性分析结果表明,灌浆期直链淀粉积累速率与AGPP、SSS、GBSS和SBE活性变化呈显著或者极显著正相关,支链淀粉和总淀粉积累速率与AGPP、SSS和SBE活性变化显著或极显著正相关。
4.在异淀粉酶作用下,支链淀粉的分支链裂解,可分为A链和短B链(DP<29),中等长度B链(2965.8)。不同长度的支链淀粉分支链相对含量在小麦籽粒发育期不断地变化,并且在糯性与非糯性小麦材料中的积累动态过程存在差异。灌浆期,AGPP、SSS、GBSS和SBE酶与支链淀粉分支链关系密切。
5.控制淀粉合成的关键酶有四大类:AGPP、GBSS、SS和SBE,相应的其编码基因也分为四类,第一类是AGPP大亚基和小亚基的编码基因AGPP-L和AGPP-S;第二类是GBSS的编码基因GBSSI;第三类是SSS的编码基因SSI、SSII和SSIII;第四类是SBE的编码基因SBEI、SBEIIa和SBEIIb。应用实时荧光定量的方法研究各淀粉合成酶编码基因在灌浆期不同时期的实时表达,结果表明:除GBSSI外,其它淀粉合成酶编码基因在两个小麦材料中的表达模式基本相似,大致为低-高-低的单峰曲线和高-低-高-低的双峰曲线模式。GBSSI在非糯性小麦中呈现双峰曲线的表达模式,而在糯性小麦中一直处于低效表达的状态。根据小麦胚乳中的各个淀粉合成酶编码基因在籽粒灌浆不同时期的表达特性,我们可将其分为四类:第一类是在整个灌浆期均高效表达,即参与种皮中临时性淀粉的合成,也参与胚乳淀粉的合成,SSII;第二类是在籽粒种皮合成初期高效表达,参与临时性淀粉的合成,SBEIIa;第三类是在胚乳淀粉合成时期高效表达,有AGPP-L、AGPP-S、GBSSI、SSIII和SBEIIb;第四类是在灌浆初期低效表达,在胚乳合成时快速增长,淀粉合成中后期恒定表达,且在胚乳淀粉合成中起着重要作用的基因,有SSI和SBEI。因此,淀粉合成酶编码基因的表达存在时序性和组织特异性,在淀粉合成过程中起着不同的调控作用
6.比较各基因在两种不同类型小麦中的表达量差异,结果表明GBSSI、SSIII在非糯性小麦中高效表达,在糯性小麦中低效表达;SBEI、SBEIIb在糯性小麦中高效表达,在非糯性小麦中低效表达。
7.通过以上结果得出,淀粉合成酶编码基因在灌浆期小麦籽粒发育过程中的表达规律是:SSI基因在灌浆中-后期恒定的表达,而SSII在早期和中期高效表达,SSIII基因在中期高效表达,GBSSI基因则在整个灌浆期高效表达;SBEIIa基因在灌浆初期-中期高效表达,SBEIIb在灌浆中期高效表达,SBEI则在灌浆中-后期高效表达。
8.以下实验结果证明支链淀粉的合成早于直链淀粉:
(1)非糯小麦在花后5天前时未检测到直链淀粉存在,而此时已经检测到支链淀粉含量,并且糯小麦仅含有支链淀粉;
(2)在灌浆早期,可以检测到SSS酶活性,而此时却没有检测到GBSS酶活性,灌浆期主要参与支链淀粉合成的SSS酶活性均大于主要参与直链淀粉合成的GBSS酶活性;
(3)用异淀粉酶水解花后5天左右的支链淀粉α-1,6糖苷键,结果证明,支链淀粉的分支链已经形成,此时无直链淀粉。
(4)淀粉合成酶编码基因在灌浆期的表达研究结果表明,在灌浆初期,SSII和SBEIIa的表达量较高,而这两个基因均属于支链淀粉合成酶编码基因,虽然GBSSI在非糯性小麦中也有表达,但是由于GBSSI属于转录后调控基因,根据在糯性小麦后期GBSSI的表达量可知,其在灌浆初期的表达量并不能合成直链淀粉。
Two different wheat varieties were used to study the dynamic changes of theaccumulations of amylose and amylopectin, starch accumulation rate, the activity changes ofkey enzymes involved in starch biosynthesis, the accumulations of different amylopectinchain length and the expression profiles of genes which encoded starch synthase in the kernelduring grain filling. The results observed were:
1. The changes of amylose and amylopectin accumulating rates of both waxy andnon-waxy wheat varieties showed a single peak curve. The accumulation courses of bothamylopectin and amylose were well fitted to the logistic equation by relating amylopectin andamylose contents against days post anthesis. The simulation parameters revealed that thehigher contents of amylopectin and amylose resulted from greater accumulation rate andmean accumulation rate, but accumulation duration probably played a less important role forthem.
2. The activity changes of the adenosine diphosphorate glucose pyrophrylase (AGPP),soluble starch synthase (SSS), Granule-bound starch synthase (GBSS) and starch branchingenzyme (SBE) were all in the pattern of a single-peak curve, and peaked at20~25days afteranthesis.
3. The accumulation rate of amylose was significantly or highly significantly correlatedwith the activities of AGPP, SSS, GBSS and SBE the accumulation rate of amylopectin wassignificantly correlated with the activity of SSS, and the accumulation rate of starch wassignificantly or highly significantly correlated with the activities of AGPP and SSS.
4. Amylopectin were de-branched with isoamylase and named by different chains A andshort B (DP<29), mid-length B (2965.8). Different amylopectinchain contents changed continually during grain development and varied differently betweenthe two types of wheat. Moreover, the AGPP, SSS, GBSS, and SBE activities exhibited aclose correlation with the different chains of amylopectin.
5. A comprehensive analysis of the transcript levels of genes which encodestarch-synthesis enzymes was fundamental for the assessment of the function of each enzymeand the regulatory mechanism for starch biosynthesis in sink organs. Using quantitativereal-time RT-PCR, an examination was made of the expression profiles of9wheat genesencoding three classes of enzymes, i.e. ADPglucose pyrophosphorylase, starch synthase, andstarch branching enzyme in developing grain, which were AGPP-L and AGPP-S encoded thelarge and small subunit of AGPP, GBSSI was encoded GBSS, SSI, SSII and SSIII wereencoded SS, SBEI, SBEIIa and SBEIIb were encoded SBE. The results showed that theexpression profiles of AGPL, AGPP-S, SSI, SSII, SSIII, SBEI, SBEIIa and SBEIIb weresimilar between non-waxy and waxy types of wheat, except GBSSI. Four patterns ofexpression in the seed were identified: group1gene, SSII, which was highly expressedthroughout granule development; group2gene, SBEIIa, which was expressed very early ingrain formation and are presumed to be involved in the construction of fundamental cellmachineries, de novo synthesis of glucan primers, and initiation of starch granules; group3genes, AGPP-L、AGPP-S, GBSSI, SSIII and SBEIIb, which are highly expressed throughoutendosperm development; group4genes, SSI and SBEI which have transcripts that are low atthe onset but which rise steeply at the start of starch synthesis in the endosperm and arethought to play essential roles in endosperm starch synthesis, Thus, The modes of geneexpression were tissueand developmental stage-specific.
6. During the whole stage of gain development, GBSSI and SSIII genes higher expressedin non-waxy while SBEI and SBEIIb were lower expressed in non-waxy wheat.
7. During the whole stage of grain development, SSI was generally expressed over themid-late grain development, while SSII was higher expressed over the early-mid graindevelopment, SSIII was higher expressed at the middle of grain development, and the GBSSIwas higher expressed during whole stage of grain development; SBEIIa was expressed overthe early-mid grain development, SBEIIb was higher expressen at the middle of graindevelopment, and SBEI was higher expressed over mid-late grain development.
8. The results showed that the beginning biosynthesis of amylopectin was earlier thanamylose, based on the results as below:
(1) The amylopectin appeared at5days after anthesis in non-waxy type of wheat;meanwhile, we did not found the amylose at5days after anthesis. The amylose content wasdetected about10days after anthesis, and the accumulation of amylopectin was increasedrapidly at this time. Moreover, we did not found amylose in the waxy type of wheat.
(2) At5days after anthesis, we detected SSS activity which mainly was involved inamylopectin synthesis, but GBSS activity which mainly was involved in amylose synthesiswas not detected.
(3) The amylopectin which were obtained at5days after anthesis were de-branched withisoamylase, we found that the different chains of amylopectin were synthsized at this time.
(4) The expression level of genes which encoded starch snthase told us that the SSII andSBEIIa, which encoded the starch synthase maily involved in amylopectin synthesis, werehigher expressed at the initiation of grain development. Even though the GBSSI wereexpressed at this time, it probably belonged to post-transcriptional control gene. According tothe espression levele of GBSSI in waxy type of wheat, we speculated that the expression levelwere too lower to amylose synthesis.
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