共转化获得无抗性标记的转反义蜡质基因籼稻

【目的】利用转基因技术将反义蜡质基因导入籼稻成熟胚愈伤组织中,改良籼稻稻米直链淀粉含量。【方法】用根癌农杆菌介导的方法将p13W8质粒（含反义蜡质基因）和pCAMBIA1300质粒（含抗潮霉素抗性基因）对2个籼稻品种的成熟胚愈伤组织进行遗传共转化。【结果】供试材料愈伤组织继代培养18～24 d后具有较高的转化频率和分化频率,可作为共转化的良好受体;抗性愈伤组织PCR检测结果表明,抗潮霉素抗性基因的阳性检测率为97.6%,反义蜡质基因的阳性检测率为27.1%。在46株转基因潮霉素抗性植株中,反义蜡质基因阳性株为5株,占转基因植株的10.9%。经PCR检测,发现上述4个株系T1群体中发生抗潮霉素抗性基因和反义蜡质基因片段的分离,从286个T1单株中筛选到了39个单株只带反义蜡质基因片段,而无潮霉素抗性基因的转基因植株,占T1单株总数的13.6%。部分转反义蜡质基因植株种子的直链淀粉含量有不同程度的降低。【结论】通过农杆菌介导的遗传共转化方法,将反义蜡质基因Waxy导入籼稻成熟胚愈伤组织,获得了直链淀粉含量明显降低的仅含反义蜡质基因籼稻株系。     

用转基因技术培育水稻软米品种

通过根癌农杆菌(Agrobacterium tumefacients)介导的方法,进行水稻反义蜡质基因的遗传转化,获得水稻转基因植株.经PCR分子检测,转基因植株均为阳性.对T2代单株的直链淀粉含量测定,获得了直链淀粉含量在14%以下的单株,经T3株系不同单株的直链淀粉含量测定得到了稳定的软米株系,证明反义蜡质基因的转化对降低稻米直链淀粉含量有效.这对稻米淀粉品质改良,丰富水稻种质资源有积极作用.     

用反义RNA技术创造高直链淀粉玉米材料

 【目的】利用反义RNA技术调控玉米淀粉的生物合成过程,创造高直链淀粉玉米材料。【方法】克隆玉米淀粉分支酶(sbe2a)基因片段,以载体pWGLL为基础,构建高效反义表达载体,通过花粉管通道法将其导入玉米自交系铁7922中。【结果】获得了4株转基因株系,GFP表达检测、PCR扩增和Southern杂交结果表明,目的基因已整合到基因组中,且能够遗传。对4株转基因植株进行RT-PCR和淀粉分支酶活性检测,结果表明转反义sbe2a玉米淀粉分子酶基因的转录受到了明显抑制,淀粉分支酶活性明显低于野生型,相差最多的降低79.4%;直链淀粉含量也发生明显的变化,最高的提高了84.3%,且总淀粉含量与对照之间基本没有差异。【结论】采用反义RNA技术通过沉默内源sbe2a,可获得高直链淀粉含量的玉米材料。     

太湖流域粳稻地方品种食味品质分析

为了充分利用太湖流域优质的粳稻种质资源,选取161份太湖流域粳稻地方品种(系)为试验材料,对其10个食味性状进行分析评价。结果表明,太湖流域不同水稻品种(系)的食味品质间存在极显著差异。不同品种的稻米RVA谱曲线特征值与直链淀粉含量、蛋白质含量、脂肪酸含量间均存在一定的相关性;崩解值和消减值2个指标不仅可以反映RVA谱曲线的基本特征,同时又可以反映稻米食味品质的优劣。低直链淀粉含量的水稻品种的RVA谱仍存在较大的差异。随着脂肪酸含量的增加,峰值黏度、热浆黏度、崩解值均极显著下降(P<0.01);而消减值则随着脂肪酸含量的增加而极显著提高(P<0.01)。因此,从RVA谱曲线特征值看,本试验选用的太湖流域水稻地方品种(系)的部分稻米食味品质优良,可作为优质高产的种质资源进行利用。     

香稻种质稻米淀粉RVA谱特征分析

采用粘度速测仪分析了42份来自不同国家的香稻种质资源的稻米淀粉粘滞性谱(RVA谱)和特征值间的相关性.结果表明,香稻种质稻米淀粉RVA谱特征值差异显著,谱线分化明显,各个特征值与稻米直链淀粉含量的高低也具有较好的相关性.因此.在测定直链淀粉含量的基础上辅以RVA谱特征,可以用来评价稻米的蒸煮与食用品质和筛选优质水稻品种.     

太湖流域水稻地方种的稻米品质RVA谱多样性

对494份太湖地区晚粳稻、糯稻、早籼稻地方种材料的稻米品质RVA谱特征进行测定与分析,结果表明:这些种质资源的稻米品质RVA谱曲线特征分化明显,种质问RVA特征值均有极显著差异,呈现出从籼性到糯性连续变化的特征,形成不同的特征类群.大多数种质(51.9%)的RVA谱的消减值在300～1 000 cp,属于典型的粳稻品种;少量种质(1.4%)具有糯性淀粉RVA谱特征;一些种质(9.8%)表现出为典型的籼稻淀粉RVA谱特征;部分种质(38.0%)具有优质的RVA谱特征,即消减值为负值,表明种质的淀粉性质存在明显的差异.各特征值的表型遗传多样性指数均大于1,其中崩解值的遗传多样性指数达2.04.RVA谱各特征值问显著相关,并可用崩解值、消减值2个指标来简化定量描述稻米RVA谱的特征.     

用RNA干扰技术创造高直链淀粉马铃薯材料

【目的】创造块茎高直链淀粉含量的转基因马铃薯材料。【方法】采用RT-PCR技术分别克隆了马铃薯Sbe 1基因CDS内300 bp的片段SⅠ和Sbe 2基因CDS内410 bp的片段SⅡ,并将SⅠ和SⅡ顺序连接得到融合片段SⅢ;以载体pHANNIBAL和pART27为基础,构建具有SⅢ反向重复结构的植物表达载体pRNAiⅢ;采用农杆菌介导法转化马铃薯优良品种陇薯3号、甘农薯2号和大西洋。【结果】获得了融合片段SⅢ,构建了以Sbe 1基因和Sbe 2基因为靶标的RNA干扰载体pRNAiⅢ,通过农杆菌介导法获得了24个转基因株系,其中21个转基因株系试管薯的淀粉粒形态发生明显变化,表观直链淀粉含量介于59.31%～87.14%,比受体材料平均高出3.2倍。RT-PCR分析表明,在8个直链淀粉含量超过80%的转基因株系中检测不到Sbe 1和Sbe 2基因mRNA的积累。【结论】采用RNAi技术通过沉默内源Sbe 1和Sbe 2,可获得高直链淀粉含量的马铃薯材料。     

不同地点南粳9108稻米直链淀粉含量及RVA谱特征值分析

以2014年、2015年分别在江苏省18个地区种植的南粳9108稻谷为材料,测定其稻米直链淀粉含量和快速黏度仪(apid visco-analyzer,简称RVA)谱特征值,对不同地点南粳9108稻米直链淀粉含量和RVA谱特征值的变异及相关性进行分析。结果表明,2年不同地点种植的南粳9108稻米直链淀粉含量的差异明显。纬度最高的东海地区其直链淀粉含量最高,纬度最低的南京地区其直链淀粉含量最低,其他地区的均在10%左右。RVA谱特征值分析结果表明,RVA谱特征值在不同地点和不同年份间也存在差异,2015年RVA谱特征值的变异系数除糊化温度外均明显高于2014年。不同地点RVA谱各特征值变化不一致,糊化温度和峰值时间基本比较稳定,差异较小,其他特征值差异均较大,消减值的变异系数最大,2014年变异系数从大到小依次为消减值、回复值、崩解值、最低黏度、最终黏度、峰值黏度、糊化温度、峰值时间,2015年的排序为消减值、最低黏度、最终黏度、崩解值、回复值、峰值黏度、峰值时间、糊化温度。2年36个地点种植的南粳9108稻米AC与RVA谱特征值的相关性分析结果表明,直链淀粉含量与糊化温度呈极显著负相关,与回复值、峰值时间呈显著正相关,与峰值黏度、最低黏度、最终黏度、消减值等呈正相关,与崩解值呈负相关,但均未达显著水平。     

板栗淀粉糊化特性与淀粉粒粒径及直链淀粉含量的关系

 【目的】分析3个品种群的33个主栽品种板栗淀粉糊化特性与直链淀粉含量及淀粉粒粒径之间的关系,为评价板栗糯性提供理论依据。【方法】采用常规理化分析方法测定板栗淀粉含量以及直链淀粉含量;用快速黏度分析仪（RVA）测定板栗淀粉糊化特性;利用S3500型激光粒度分布仪测定淀粉粒粒径分布;使用SPSS软件对数据进行差异显著性检验和聚类分析。【结果】不同品种板栗淀粉的RVA谱特征值之间存在较大差异,北方品种群板栗淀粉的峰值黏度、谷值黏度、最终黏度和稀懈值的总体水平均较其它两个品种群板栗高,各品种群之间差异显著;北方品种群的回冷值、峰值时间和糊化温度最低。板栗淀粉的糊化特性具有较明显的地域分布特征。不同品种板栗的总淀粉含量和直链淀粉含量均存在一定差异,北方品种群板栗的总淀粉含量显著低于其它两个品种群,南方品种群板栗淀粉中直链淀粉含量显著高于其它两个品种群。不同品种板栗粒径之间存在显著差异,淀粉粒粒径与品种密切相关,同时也具有较明显的地域分布特征。【结论】聚类分析将33个品种板栗分为4大类,北方品种群板栗普遍具有低直链淀粉含量、高峰值黏度的特点;南方品种群中的大部分板栗品种具有高直链淀粉含量和低峰值黏度的特点;中间类型品种群中一部分品种具有近于北方品种群板栗的特点,一部分具有近于南方品种群板栗的特点。板栗的直链淀粉含量和糊化峰值黏度可以作为衡量板栗糯性品质的指标。     

灌浆期表观直链淀粉含量相似品种稻米胶稠度和RVA谱的动态差异

 【目的】从品质形成的动态角度探讨相似AAC（apparent amylose content, 表观直链淀粉含量）水稻品种其米质动态形成的差异。【方法】选用成熟种子表观直链淀粉含量相似而米质不同的两个水稻品种武育粳3号和30优917为材料,研究其灌浆过程中直链淀粉含量、胶稠度及RVA谱动态变化差异。【结果】两个品种在籽粒灌浆过程中直链淀粉含量、胶稠度和RVA谱的动态变化存在显著差异。在花后5—15 d2个品种的直链淀粉含量存在显著差异,到灌浆后期则表现相近。胶稠度和RVA谱在籽粒灌浆的全过程中均有明显差异。与30优917相比,武育粳3号的胶稠度在花后5—10 d较软,到籽粒灌浆的中后期（花后20—40 d）却明显比30优917硬;用RVA测得的武育粳3号粘性较高,表现为较高的崩解值、较小的正消减值和较低的成糊温度。【结论】相似AAC水稻米质间的差异表现在灌浆期米质性状的动态形成存在显著差异,口感较好的武育粳3号其米粒形成过程中有较高的最高黏度、崩解值和较小的正消减值。     

Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice (Oryza sativa L.ssp.japonica)

Anther culture pure lines with antisense Wx gene were generated by Agrobacterium tumefaciens-mediated co-transformation followed by anther culture in transgenic rice (Oryza sativa L.ssp.japonica).The antisense Wx transgenic pure lines were used to analyze the differences and the relationship between RVA eigenvalues among the lines with different amylose contents.77 pure lines of anther culture having antisense Wx gene were studied for the amylose content of its offspring cultivar,Wuyunjing 7.It was found that levels of amylose content were similar to the control in 19 lines (16.0%);1-5%lower than control in 50 lines,of which,within 11.0-13.9% in 40 lines;and 3.1-4.0% in 8 lines.The effect of the amylose content on RVA eigenvalue was analyzed through the RVA profile graphic comparison and test of significance for RVA eigenvalues.The analysis of RVA profiles with different amylose contents indicated that there were two kinds of RVA profiles within genetically similar cultivars.The lines with 3.1-4.0% of amylose content were distinctly different from other lines and conventional glutinous rice.The comparison of the similarity curve of RVA profile of the lines with different amylose contents showed that the lines with lower amylose content had a distinguished RVA profile with the curve increasing gradually,but not exceeding the first apex.The test of significance for eigenvalues of the RVA profile in the lines with different amylose contents indicated that there were five eigenvalues remarkably different among the lines with 3.1-4.0% of amylose content compared with other two groups.Further,there were three eigenvalues remarkably different among the lines with 11.0-13.5% of amylose content compared with control group.An optimized mathematical model,which characterizes the relationships between the amylose content and RVA eigenvalue,has been established in this study.From this model,it was clearly indicated that the parameters PeT and SBV were more related to amylose content.It was concluded that the differences in amylose content not only affected the eigenvalues of RVA but also caused different RVA profiles within genetically similar cultivars.The introduction of antisense Wx gene could lead to reduce the amylose content and lay theoretical foundation for quality improvement in rice breeding programs.     

Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice(Oryza sativa L.ssp.japonica)

Anther culture pure lines with antisense Wx gene were generated by Agrobacterium tumefaciens-mediated co-transformation followed by anther culture in transgenic rice(Oryza sativa L.ssp.japonica).The antisense Wx transgenic pure lines were used to analyze the differences and the relationship between RVA eigenvalues among the lines with different amylose contents.77 pure lines of anther culture having antisense Wx gene were studied for the amylose content of its offspring cultivar,Wuyunjing 7.It was found that levels of amylose content were similar to the control in 19 lines(16.0%);1-5% lower than control in 50 lines,of which,within 11.0-13.9% in 40 lines;and 3.1-4.0% in 8 lines.The effect of the amylose content on RVA eigenvalue was analyzed through the RVA profile graphic comparison and test of significance for RVA eigenvalues.The analysis of RVA profiles with different amylose contents indicated that there were two kinds of RVA profiles within genetically similar cultivars.The lines with 3.1-4.0% of amylose content were distinctly different from other lines and conventional glutinous rice.The comparison of the similarity curve of RVA profile of the lines with different amylose contents showed that the lines with lower amylose content had a distinguished RVA profile with the curve increasing gradually,but not exceeding the first apex.The test of significance for eigenvalues of the RVA profile in the lines with different amylose contents indicated that there were five eigenvalues remarkably different among the lines with 3.1-4.0% of amylose content compared with other two groups.Further,there were three eigenvalues remarkably different among the lines with 11.0-13.5% of amylose content compared with control group.An optimized mathematical model,which characterizes the relationships between the amylose content and RVA eigenvalue,has been established in this study.From this model,it was clearly indicated that the parameters PeT and SBV were more related to amylose content.It was concluded that the differences in amylose content not only affected the eigenvalues of RVA but also caused different RVA profiles within genetically similar cultivars.The introduction of antisense Wx gene could lead to reduce the amylose content and lay theoretical foundation for quality improvement in rice breeding programs.     

Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice （Oryza safiva L. ssp. japonica）

Anther culture pure lines with antisense Wx gene were generated by Agrobacterium tumefaciens-mediated co-transformation followed by anther culture in transgenic rice （Oryza sativa L. ssp. japonica）. The antisense Wx transgenic pure lines were used to analyze the differences and the relationship between RVA eigenvalues among the lines with different amylose contents. 77 pure lines of anther culture having antisense Wx gene were studied for the amylose content of its offspring cultivar, Wuyunjing 7. It was found that levels ofamylose content were similar to the control in 19 lines （16.0%）; 1-5% lower than control in 50 lines, of which, within 11.0-13.9% in 40 lines; and 3.1-4.0% in 8 lines. The effect of the amylose content on RVA eigenvalue was analyzed through the RVA profile graphic comparison and test of significance for RVA eigenvalues. The analysis of RVA profiles with different amylose contents indicated that there were two kinds of RVA profiles within genetically similar cultivars. The lines with 3.1-4.0% of amylose content were distinctly different from other lines and conventional glutinous rice. The comparison of the similarity curve of RVA profile of the lines with different amylose contents showed that the lines with lower amylose content had a distinguished RVA profile with the curve increasing gradually, but not exceeding the first apex. The test of significance for eigenvalues of the RVA profile in the lines with different amylose contents indicated that there were five eigenvalues remarkably different among the lines with 3.1-4.0% of amylose content compared with other two groups. Further, there were three eigenvalues remarkably different among the lines with 11.0-13.5% of amylose content compared with control group. An optimized mathematical model, which characterizes the relationships between the amylose content and RVA eigenvalue, has been established in this study. From this model, it was clearly indicated that the parameters PeT and SBV were more related to amylose content. It was concluded that the differences in amylose content not only affected the eigenvalues of RVA but also caused different RVA profiles within genetically similar cultivars. The introduction of antisense Wx gene could lead to reduce the amylose content and lay theoretical foundation for quality improvement in rice breeding programs.     

直链淀粉含量与稻米品质主要性状及米饭质地关系的研究

以反义Wx基因的转化系及其受体亲本武运粳7号为材料,研究遗传背景相同仅直链淀粉含量不同的4个品系主要品质性状及米饭质地的变化。结果表明：直链淀粉含量不同的4个供试品系的主要品质性状指标与米饭质地指标之间有着明显的差异。直链淀粉含量的降低使得胶稠度变软,粗蛋白含量减少,过低的直链淀粉含量会导致米粒外观呈现蜡质。稻米RVA谱特征和米饭质地明显受到直链淀粉含量的控制,直链淀粉含量明显影响稻米糊化过程中黏度的变化,高直链淀粉品系具有较高的消减值、回复值、热浆黏度及最终黏度,米粉的回生性大,米粉难以糊化,其米饭质地过硬,咀嚼有渣感,无弹性;相反,低直链淀粉含量的品系具有较低的消减值、回复值、热浆黏度及最终黏度,冷却过程中米粉的回生性小,其米饭质地较黏,咀嚼无渣感,有弹性;直链淀粉含量与最高黏度值和崩解值间的关系并非是简单的线性关系,当直链淀粉含量较低时,随着直链淀粉含量的增加,最高黏度和崩解值随之升高,而当直链淀粉含量高于一定量时,直链淀粉的增加会降低最高黏度和崩解值。     

高抗性淀粉粳稻新品系稻米淀粉链长分布与主要品质特征差异

 【目的】通过对水稻抗性淀粉性状的遗传研究,为高抗性淀粉水稻资源创新与育种提供理论和实践依据。【方法】以抗性淀粉含量有显著差异的粳稻品系为材料,比较分析高低抗性淀粉粳稻品系类型间的支链淀粉链长分布、稻米RVA谱特性值、膳食纤维和直链淀粉含量等主要品质特性的差异及其相关性。【结果】高低抗性淀粉水稻品系间支链淀粉链长分布差异显著,高抗性淀粉水稻品系的支链淀粉中短链部分即聚合度为5～12所占的相对百分比率显著低于低抗性淀粉类型品系,而长链尤其是聚合度为13以上的长链比率明显高于低抗性淀粉类型品系。膳食纤维和直链淀粉含量,高抗性淀粉品系显著高于低抗性淀粉品系,最高黏度值、最终黏度值、热浆黏度值和崩解值等,高抗性淀粉品系均显著或极显著低于低抗性淀粉类型品系。抗性淀粉含量与支链淀粉短链聚合度（DP≤12）呈极显著负相关,但与支链淀粉的中长链聚合度（12＜DP≤36）呈极显著正相关,与直链淀粉含量和膳食纤维含量呈极显著正相关。【结论】水稻中支链淀粉的链长分布、直链淀粉含量和稻米RVA谱特性值与稻米抗性淀粉含量关系密切,在育种实践中可作为育种选择指标之一。     

水稻栽培品种淀粉合成相关基因来源及其对品质的影响

【目的】明确高产水稻品种中与淀粉合成相关基因的性质，为稻米品质改良提供指导。【方法】以53个典型的籼粳品种和近年育成的高产水稻品种为材料，分析了供试品种的理化品质和RVA谱特征，并利用根据籼粳基因组序列差异设计的Wx、Sbe1、Sbe3基因的分子标记，检测了53个水稻品种的基因型，并分析了3个基因位点的遗传学效应。【结果】3个分子标记均能很好的区分3个位点上等位基因的籼粳来源，根据3个位点的基因型可将53个品种分为6种类型。单个基因遗传效应分析表明：在不同基因型品种间淀粉的理化特性（AC，GC, RVA）存在显著或极显著差异，3个基因的联合效应在不同基因型组合间也存在显著的差异。【结论】3个基因在高产品种培育过程中得到了广泛的交流和重组，而且3个基因位点上的不同等位基因（籼粳）对稻米淀粉的合成具有不同的遗传效应。其中Wx基因的影响是最主要的，Sbe1、Sbe3基因次之。3个基因位点的分子标记可为高产品种的品质改良提供快捷的工具。     

糯小麦与其它作物淀粉特性的比较研究

【目的】阐明糯小麦与其它作物的淀粉理化特性差异,分析糯小麦淀粉在食品工业中的应用前景。【方法】以淀粉厂批量生产的糯小麦淀粉及市售食品级马铃薯、木薯、糯玉米、红薯、普通小麦、普通玉米作物淀粉为材料,比较研究直链淀粉含量、膨胀势、淀粉色泽、透光率、快速黏度测定仪（RVA）测定的淀粉糊化特性、差示扫描量热仪（DSC）测定的热力学特性及冻融析水率。【结果】糯小麦和糯玉米直链淀粉含量均小于1%,其它作物淀粉直链淀粉含量在22%—27%;糯小麦淀粉透光率与红薯淀粉差异不显著,显著低于马铃薯、木薯和糯玉米淀粉,显著高于小麦和玉米淀粉;糯小麦淀粉膨胀势和峰值黏度与糯玉米、红薯淀粉无显著差异,显著低于马铃薯、木薯淀粉,显著高于小麦和玉米淀粉;糯小麦淀粉糊化温度（RVA测定）、起始温度（DSC测定）显著低于其它作物淀粉;糯小麦淀粉回生值与糯玉米无显著差异,显著低于其余几种作物淀粉;糯小麦淀粉冻融析水率最低,显著低于其它作物淀粉。【结论】糯小麦淀粉与常用作物淀粉的多数理化特性存在显著差异。综合比较,糯小麦淀粉糊化温度（RVA测定）低、起始温度（DSC测定）低、回生老化程度小、冻融稳定性好,优于其它作物淀粉,对于改善速溶、快餐、冷冻食品品质及延长食品货架期有显著优势。     

普通小麦SGP-A1缺失对淀粉品质的影响#br#

【目的】通过对国内外138份农家种和黑小麦材料进行SGP-A1缺失鉴定,并研究其对直链淀粉和支链淀粉含量,以及面粉糊化特性的影响,为小麦品质改良提供新种质及理论依据。【方法】采用SDS-PAGE电泳检测SGP-A1缺失情况,利用双光束双波长法测定面粉中直链和支链淀粉含量,用快速粘度测定仪(rapid viscosity analyzer,RVA)测定面粉糊化特性。【结果】供试材料中缺失SGP-A1的有26份,SGP-A1缺失显著降低籽粒中支链淀粉含量,提高直链淀粉的相对含量,并降低面粉糊化参数反弹值,但对粒重、淀粉总量及其它面粉糊化参数没有显著影响。【结论】SGP-A1缺失显著提高籽粒中直链淀粉相对含量,降低反弹值。