颗粒结合型淀粉合成酶研究进展

本文综述了小麦胚乳中合成直链淀粉的关键酶——颗粒结合型淀粉合成酶(GBSS)的研究进展，展望了GBSS的研究前景。小麦中的GBSS基因在种子中特异表达；控制3个Waxy，位点的基因Wx—A1、Wx—B1和Wx—D1，分别位于7AS、4AL和7DS上；六倍体小麦和二倍体小麦中3个GBSS的DNA序列从起始密码子到终止密码子，Wx—A1为2781bp，Wx-B1为2794bp，Wx—D1为2862bp。基因的完全编码区含有11个外显子和10个内含子，在核苷酸序列上三个糯基因彼此之间有惊人的同源性；研究表明，直链淀粉含量和淀粉糊化特性受Wx—B1的影响最大，其次是Wx-D1缺失蛋白，Wx-A1缺失蛋白的影响最小。GBSS的鉴定技术包括I2—KI染色、电泳和分子标记技术鉴定。     

宁夏春小麦种质资源Wx基因分子鉴定及其分布

为明确宁夏春小麦种质资源Waxy基因的分布情况,利用Wx-A1、Wx-B1和Wx-D1位点的6个STS标记(PA1、PA2、PB1、PB2、PD1、PD2),对299个宁夏春小麦种质资源进行等位变异检测.结果 表明,供试小麦种质的Waxy基因组成以野生型(Wx-A1a/Wx-B1a/Wx-D1a)为主,占参试材料总数的...     

小麦Wx-A1和Wx-D1位点的PCR分子标记

为了获得筛选低直链淀粉含量的分子标记,根据已知小麦Waxy基因序列设计PCR引物,通过在基因水平和蛋白质水平进行分离群体验证,建立分别专一检测Wx-A1位点和Wx-D1位点的PCR标记MAG264和MAG269。在分离群体中,MAG264标记能清楚地区分Wx-A1位点的三种不同基因型,呈共显性遗传。MAG269标记引物在野生型中扩增片段为1 400 bp,在突变型(Wx-D1bWx-D1b)中的扩增片段为800bp。但在分离群体中没有出现杂合体带型,从而使本应该表现为共显性的标记表现为显性遗传。MAG264和MAG269引物对DNA的扩增稳定性、重复性好。这些结果表明,本研究获得的Waxy基因分子标记可以用于小麦Waxy基因缺失类型的鉴定和糯质专用小麦的分子标记辅助选育。     

小麦糯性基因多重PCR体系的建立

为建立通过一次反应能够同时鉴定3个小麦糯性基因(Wx-A1、Wx-B1和Wx-D1)的多重PCR反应体系,研究了PCR反应组分和循环参数对多重PCR反应结果的影响.结果表明,反应体系中的引物浓度比例和反应的Tm值是多重PCR成功的关键,当引物浓度比例(SSR/MAG267)为1(0.1 μmol/L∶0.1 μmol/L)～1.22(0.11 μmol/L∶0.09 μmol/L)、Tm为58℃时,Wx-A1、Wx-B1、Wx-D1基因的多重PCR结果最好.该多重PCR方法能快速高效地鉴定3个Wx基因,可用于糯麦选育和小麦品质育种的复合分子标记辅助选择.     

硬粒小麦缺失4A蜡质蛋白对直链淀粉含量及淀粉品质特性的影响

为了明确硬粒小麦缺失不同Wx蛋白对直链淀粉含量的效应和Wx-B1 缺失型(缺失4A蜡质蛋白)的淀粉特性,利用SDS-PAGE电泳分析技术对1个硬粒小麦F2分离群体(来源于以硬粒小麦品系B413Wx-B1缺失型为母本、PI435045Wx-A1缺失型为父本的杂交组合)的102个单籽粒进行了Wx蛋白的筛选,并测定了其中72个单籽粒(其中Wx-B1 缺失型21粒,Wx-A1 缺失型10粒,正常型41粒)的直链淀粉含量;同时测定了11个Wx-B1 缺失型和7个正常型硬粒小麦品系面粉的直链淀粉含量、膨胀势和RVA黏度.结果表明,硬粒小麦2对Wx基因的分离符合9∶3∶3∶1的分离模式,Wx-B1 缺失型籽粒的直链淀粉含量比正常型低4.78%.Wx-B1 缺失型的面粉直链淀粉含量比正常型低5.76%,RVA高峰黏度和膨胀势比正常型分别高116.02 RVU和3.69 g/g.相关分析表明,直链淀粉含量与RVA高峰黏度和膨胀势均呈极显著负相关.     

利用Wx基因的分子标记筛选部分糯小麦的研究

为了筛选出更多用于培育优质面条小麦的Wx基因缺失材料,用分子标记技术对260份中国核心小麦种质资源进行了分析,并用SDS-PAGE进行了验证.筛选出缺失Wx-A1蛋白亚基的品种2份,缺失Wx-B1蛋白亚基的品种14份,得到1份不同于"白火麦"的Wx-D1蛋白亚基缺失的地方品种"秃芒麦".对它们的地区分布分析发现,长江以北各麦区的小麦品种Wx基因缺失多于长江以南各麦区,且分布更为集中.对收集的20对Wx基因的分子标记引物进行了筛选,并对PCR扩增条件和产物分析条件进行优化,得到了3对可以在相同条件下对Wx基因进行筛选的引物,且能用同一电泳条件对产物进行分析,提高了分析效率.     

糯小麦Wx基因的遗传特性

为了明确糯小麦杂交后代肌基因的分配规律及糯性籽粒出现的频率，以同时缺失Wx-A1、Wx-B1和Wx-D1基因的H9908、EH为纯糯亲本，普通小麦扬麦158、苏麦6号、济南17和豫麦50为非糯亲本，配制了16个(8对正反交)杂交组合，采用0．2％ I2 2％KI碘液对杂种F1单株籽粒剖面染色，鉴定了糯性籽粒的分离特点。结果表明，在16个组合间糯性籽粒出现的频率为1．20％～1．61％，非糯与全糯的比例符合63：1的理论比例；独立性测验表明，糯性籽粒在8对正反交组合问出现的比例差异不显著，全糯质与非糯质基因紧密连锁。文中还对糯小麦品种的选育作了讨论。     

Waxy蛋白缺失小麦及正反交组合淀粉特性的研究

研究了Waxy蛋白缺失类型小麦材料及正反交组合F1代的直链淀粉含量、膨胀势和降落数值等淀粉理化特性以及它们之间的相关性。结果表明：降落数值与直链淀粉含量呈正相关;膨胀势与直链淀粉含量无相关;正、反交组合F1代种子的直链淀粉含量介于糯麦和普通小麦两类亲本之间,为中亲效应。反交组合F1代比正交F1代直链淀粉含量高,说明小麦的糯性遗传为细胞质遗传。正、反交组合的降落数值和膨胀势差异均不显著。说明这两个性状的遗传为非胞质遗传。     

中国大麦种质资源Wx蛋白分析及糯大麦分子标记验证

利用1D-SDS-PAGE分析了中国167份不同直链淀粉含量类型的大麦种质资源Waxy 蛋白类型,发现在低直糯大麦和无直糯大麦两种类型中,可能存在四种生化遗传变异类型。其中有两种与无直糯大麦有关,一种是Wx基因可以正常转录,但转录产生的Wx蛋白无活性,不能催化直链淀粉合成;另一种是Wx基因不能正常转录,既无Wx蛋白产生,也无直链淀粉合成。另外两种与低直糯大麦有关,一种是Wx基因可以正常转录,但转录产生的Wx蛋白活性有限,催化直链淀粉合成的功能降低;另一种是Wx基因不完全正常转录,产生的Wx蛋白数量有限,影响直链淀粉合成。利用4个大麦与小麦的STS标记和2个大麦与小麦的SSR标记对不同Wx蛋白类型进行糯性鉴定,结果表明,迄今利用的Wx基因的STS引物P1可能只适合用于CDC Candle遗传类型相同的糯大麦品种或基因型的鉴定。其它标记均不能用于鉴定大麦糯性胚乳表型,因此当前对于糯大麦育种及Wx分子鉴定研究而言,还有待于开发新的分子标记。     

面条品质性状相关基因在四川小麦中的分布

为给面条小麦亲本材料的选择提供依据,利用多重PCR技术分析了Wx-B1和2个主效PPO位点的等位基因在104份四川主栽小麦和骨干亲本中的分布,并检测了59份新育成品系的淀粉糊化特性.结果表明,控制低PPO活性的等位基因PPO-2Ab和PPO-2Da的频率分别为42.3%和83.6%,Wx-B1蛋白缺失基因Wx-B1b的频率为26.9%,四川小麦淀粉特性整体表现较优良.Wx-B1缺失小麦在峰值粘度、稀懈值、最终粘度和反弹值方面与正常类型间差异显著.绵阳26 、川麦107、川麦43等是面条小麦育种的优良亲本.准确、实用、高效多重PCR技术适合于多位点控制的品质性状的标记辅助选择.     

小麦Wx-A1和Wx-B1亚基的生化与分子标记检测

为了解小麦品种(系)Wx亚基的缺失类型,为品质育种提供依据,利用SDS-PAGE和STS分子标记技术对99份国内外小麦品种(系)Wx-A1和Wx-B1亚基的缺失型进行检测.结果表明,供试小麦品种(系)中Wx-B1亚基缺失的有19份,未检测出Wx-A1亚基缺失材料.STS标记结果中,Wx-7A位点的显性标记引物在野生型中扩增出一条1 172 bp的特异带,即Wx-A1a基因出现;Wx-7A位点的共显性标记引物在野生型中扩增出一条593 bp的特异带,在缺失类型中扩增出一条574 bp的特异带.Wx-4A位点的显性标记引物在野生型中扩增出一条425 bp的特异带,即Wx-B1a亚基出现,而在缺失该亚基的突变材料中没有扩增出该特异带.     

一些小麦品种的淀粉特性分析

为了筛选出淀粉品质优良的面条小麦品种,分析了200份国内小麦品种(系)和6份澳大利亚小麦品种的淀粉特性及Waxy蛋白亚基组成.与国外优质面条小麦相比,国内小麦的淀粉品质总体上有一定差距,但主要淀粉特性的变异范围比较广,如直链淀粉含量变幅为17.79%～27.14%,膨胀势为3.77～10.45g/g,峰值粘度为491.50～4 497.00 cP,低谷粘度为74.00～3 042.00 cP,崩解值为584.50～1 986.50 cP.国内小麦品种(系)中有15份缺失Wx-B1蛋白亚基,其直链淀粉含量、峰值粘度和崩解值的平均值优于正常类型品种(系),如花培18-5直链淀粉含量低于19%,峰值粘度高于4000 cP,崩解值高于1 500 cP.河南省小麦品种(系)的淀粉特性优于其他省市.     

青海高原春小麦糯性新品系的筛选及其直链淀粉含量

为了培育适合青海高原种植的春小麦糯性新品系,利用DNA分子标记技术检测了80株青春533×糯麦1号F2代材料的Wx基因缺失情况,同时测定了不同Wx基因缺失类型F2代材料的小麦籽粒直链淀粉含量。结果表明,在80株F2代分离群体材料中,单缺失WxA1或WxB1或WxD1基因的材料分别为8份、11份、9份;同时缺失WxA1和WxD1基因的材料为10份,同时缺失WxB1和WxD1基因的材料为12份,同时缺失WxA1和WxB1基因的材料为9份;WxA1、WxB1和WxD1基因全部缺失的材料为8份。Wx基因的缺失数量与直链淀粉含量的高低有密切关系,缺失1个Wx基因（部分糯小麦）,直链淀粉含量比对照（青春533）降低5.28%~11.35%;缺失2个Wx基因（部分糯小麦）,直链淀粉含量比对照降低10.73%~19.81%;缺失3个基因（糯小麦）的直链淀粉含量为0.97%,比对照降低30.53%。高原春小麦与糯性小麦杂交的F2代直链淀粉含量下降幅度要比前人所研究的材料下降幅度大,并且WxB1单缺失引起直链淀粉含量下降（11.35%）比WxA1和WxD1双缺失所引起的下降幅度要大（10.73%）。     

Analyses of albumins, globulins and amphiphilic proteins by proteomic approach give new insights on waxy wheat starch metabolism

Starch is composed of two types of glucose polymers: amylose and amylopectin. The Waxy (Wx) locus controls amylose synthesis in the wheat kernel. Hexaploid wheat has three Wx loci located on chromosomes 7A (Wx-A1), 4A (Wx-B1), and 7D (Wx-D1). Eight near isogenic lines (NILs) of Triticum aestivum cv. Tremie with one, two or three Wx null alleles were used. The albumin–globulin fraction, and amphiphilic proteins were separated using 2-dimensional electrophoresis (2DE) allowing the changes in the waxy kernel to be identified. Albumin–globulin fraction showed overexpression of sucrose synthases in the waxy NILs compared to the normal form of Tremie and a decrease in many proteins related to stress and defence metabolism such as serpins. A subunit of ADP-glucose pyrophosphorylase (AGPase), which is known to play a major role in starch synthesis, was also shown to be down regulated in the waxy NILs. Amphiphilic proteins confirmed the observations made on the albumin–globulin fraction with a decrease in a stress-related protein. These different regulations linked to observations made on wheat kernel (thousand kernel weight (TKW), protein amount per grain, size and distribution of the starch granules) led to formulation of the hypothesis that waxy endosperm does not reach maturity of the wild-type endosperm.     

A novel enzyme-linked immunosorbent assay for the detection of Wx-B1 null wheat lines using a monoclonal antibody and its application for the detection of marker heterogeneity within commercial cultivars

A monoclonal antibody was raised with specificity for the granule bound starch synthase (GBSS1) Wx-B1 homeoallele of wheat (Triticum aestivum L.) using a synthetic peptide immunogen. This was used to develop a simplified enzyme-linked immunosorbent assay (ELISA) for the discrimination of Wx-B1a and Wx-B1b alleles which differ in the starch properties they confer. Discrimination of these alleles is important for the selection of wheat lines for Udon-style noodle production. The simplified ELISA worked in a single antibody (indirect) format and gave improved ease of use, discrimination of alleles and resolution relative to a previously developed 2-antibody (sandwich) ELISA. When the test was validated using breeders seed of a panel of commercial cultivars, heterogeneity of Wx-B1 alleles was observed for a significant proportion of the cultivars tested and this was confirmed using PCR analysis of the Wx-B1 and Wx-A1 genes. This observation has implications for cultivar wheat quality assessment, the application of molecular markers for variety identification purposes and the establishment of mapping populations from commercial cultivars.     

Molecular characterization of two novel null waxy alleles in Mexican bread wheat landraces

Starch is composed of two glucose polymers: amylose, formed by long linear chains, and amylopectin, which is larger and highly branched. The ratio between the two polymers (usually 22–35% amylose, 68–75% amylopectin) affects the starch properties, thereby determining different aspects of wheat quality. Waxy protein, which is the sole enzyme responsible for amylose synthesis, showed polymorphism in a collection of one hundred and three Mexican landraces, including null alleles at Wx-A1 and Wx-B1 loci, respectively. Molecular characterization of these alleles showed that the null Wx-A1 allele presented in only one accession was novel (Wx-A1o) and involved a deletion spanning the three and a half last exons of the gene. Some of the accessions lacking the Wx-B1 protein contained the common allele Wx-B1b (12.6%) (deletion of approximately 67 kb), while four others (3.8%) possessed the novel Wx-B1l, characterized by the deletion of one cytosine in the second exon of the gene and leading to a change in the ORF. This novel allele is particularly interesting because the absence of the Wx-B1 protein in this case was not associated with the lack of the other genes included in the approximately 67 kb region lost with the common null allele Wx-B1b.     

Functionality of chemically modified wild-type,partial waxy and waxy starches from tetraploid wheats

Partial waxy (reduced-amylose) and fully waxy (amylose-free) tetraploid durum wheats (Triticum turgidum L. var durum) were developed by introgression of null alleles at the Wx-A1 and Wx-B1 loci from common hexaploid wheat (Triticum aestivum L.). Purified starches were obtained from each genotype, and chemically modified by: 1) cross-linking with phosphorus oxychloride, 2) substitution with propylene oxide, and 3) sequential cross-linking with phosphorus oxychloride followed by substitution with propylene oxide. Functional properties were compared to blends of waxy and wild-type durum starches of known amylose contents. Significant differences in functionality were observed amongst the genotypes and blends after each modification. Waxy (0% amylose) and wild-type (30% amylose) typically were at the extremes of the observed ranges of functional properties. In general, the functional properties of the chemically modified starches were dependent upon amylose content. Starches from Wx-B1 null lines (24% amylose), were an exception. After substitution, such starches had the significantly highest value for RVA final viscosity, and generally performed in a manner similar to starch blends of 12–18% amylose.