运用农杆菌介导法将PTA和反义Wx双基因导入水稻的研究

利用根癌农杆菌将含多基因(hyg选择标记基因、反义Wx基因、PTA基因、GFP和GUS报告基因)的pCAMBIA1304载体导入水稻品种(501R、中花9号和日本晴)的幼胚愈伤组织,分别在含35mg/L、45mg/L和65mg/L潮霉素浓度的筛选培养基上筛选获得抗性愈伤。后经PCR检测,从415株T0代再生植株中选出92株(其中501R、中花9号和日本晴分别为4株、43株和45株)。对这些转基因后代植株进行Southernblotting分析表明:hpt、反义Wx和PTA基因已经整合进植物基因组中,绝大多数转基因植株后代的表型正常。成熟种子直链淀粉含量分析表明,部分转基因植株的T1代种子中的直链淀粉含量有部分程度的下降,最低的已下降至6.3%,与对照相比下降了9.8%。     

水稻蜡质基因(Wx)反义片段在转基因后代中的遗传稳定性

用根癌农杆菌(Agrobacterium tumefaciens)介导的遗传转化方法，将携带水稻蜡质基因反义片段导入中花11号粳稻中，分析了20个独立转化的转基因植株。经GUS染色、PCR分子检测，证明蜡质基因反义片段已整合到这些植株的染色体组中；在转基因植株后代T1的籽粒外观糯性检测中，有8个转基因植株检测到糯性籽粒的存在；在12个转基因植株T2后代直链淀粉含量测定中，有5个T3的直链淀粉含量比对照有明显的降低；在T3、T4的直链淀粉含量测定中，获得了直链淀粉含量比对照低1％的株系；来自T2的糯稻株系其后代能相对稳定遗传。在籽粒GUS检测和外观糯性检测中，发现二者的分离比存在差异。     

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.     

转反义 Wx基因糯稻的显性遗传及对稻米粒重的效应分析

 利用直链淀粉含量下降至 2 %左右的 3个转反义Wx基因糯稻为研究材料 ,较为系统地分析了转基因糯稻的遗传及其直链淀粉含量下降后对于稻米粒重的影响。结果表明由转基因技术获得的糯性性状与其它转基因性状一样 ,表现为显性遗传 ,这与一般的糯性变异不同。导入反义Wx基因使直链淀粉含量从 17%下降至糯稻水平时 ,其稻米粒重相应降低 6 %～ 8% ,且下降幅度与直链淀粉含量的下降幅度呈显著正相关。小区试验结果表明 ,由此获得的转基因糯稻仍具有原受体亲本的高产潜力 ,利用转基因技术培育既高产又优质的水稻品种 ,尤其是高产糯稻新品种是切实可行的     

去除标记基因的水稻转基因研究

以p1300(含潮霉素抗性标记基因)和p13W8(含反义蜡质基因)为供体DNA，通过根癌农杆菌(Agrobacterium tumefaciens)介导的方法，对水稻品种寒丰B的幼胚愈伤组织进行共转化，获得35份转基因植株。经PCR检测，有5份检测到反义蜡质基因和潮霉素抗性基因共存。在其T1代材料中，通过PCR检测获得24份只带有反义蜡质基因而无潮霉素标记基因的转基因植株。结果证明：在共转化植株后代中，通过筛选可获得无抗性标记基因的水稻转基因植株。     

农杆菌介导法获得转可溶性淀粉合成酶基因籼稻

采用农杆菌介导法将胚乳特异性表达谷蛋白1(Glutelinl，Gtl)基因控制下的可溶性淀粉合成酶基因sss导入籼稻恢复系明恢86，共获得53个独立转化再生植株。PCR检测表明，sss基因已整合进水稻的基因组中。直链淀粉含量测定结果表明，转基因植株后代直链淀粉含量较对照有较大幅度的下降。     

陕西省水稻Wx基因种质资源遗传多样性分析

稻米品质与稻米中直链淀粉含量(AC)有关,而直链淀粉含量与蜡质基因(Wx)的基因型有关,研究表明Wx基因第一内含子的剪接效率决定直链淀粉含量。明确陕西省主要水稻种质资源中Wx基因的基因型,可为水稻品质育种提供依据。应用CAPS法对112份陕西省水稻主要种质资源Wx基因第一内含子供体+1位碱基G/T进行检测,根据G/T碱基将112份供试材料的Wx基因分为GG、GT、TT等3种基因型。同时,按国标检测3种不同G/T型37份稻米的直链淀粉含量及其他相关性状。结果显示,112份供试材料中有1份材料为GG型,约占1%;25份为GT型,占22%;86份材料为TT型,约占77%。说明Wx基因第一内含子供体+1位碱基G/T的多态性与AC含量有良好的对应关系,与垩白粒率和垩白度有一定的对应关系。     

杂交水稻骨干亲本Wx基因第一内含子+1位碱基多态性分析

【目的】直链淀粉含量是影响稻米品质的重要因素,而编码颗粒结合型淀粉合成酶(granule-bound starch synthetase,GBSS)的蜡质基因(Wx)是影响直链淀粉含量的主效基因。本研究拟分析120份杂交水稻亲本的Wx基因第一内含子+1位碱基的多态性,以期研究其对内含子的剪接效率及Wx的表达的影响,为亲本育种筛选提供理论参考。【方法】选取120份水稻亲本材料,采用CTAB法提取基因组DNA,并以此为模版PCR扩增Wx基因片段;采用限制性内切酶Acc I对PCR产物进行酶切分析,根据琼脂糖电泳结果分析Wx基因第一内含子+1位碱基类型;同时测定其中19份亲本的直链淀粉含量、胶稠度和碱消值。【结果】PCR扩增结果显示所有材料中均可扩增出清晰且单一的目的条带;PCR产物的Acc I酶切分析结果显示,其中81份亲本的Wx基因第一内含子+1位碱基是T,即为T型,占总数的67.5%,38份亲本为G型,占总数的31.67%,而仅有1份亲本为杂合型,即GT型,占总数的0.83%。19份亲本的直链淀粉含量测定结果表明,7份G型亲本的直链淀粉含量比12份T型亲本的高,且其中6个G型亲本的直链淀粉含量平均值均超过20%。另外,测定结果表明T型亲本和G型亲本的胶稠度和碱消值差别不明显。【结论】120份杂交水稻骨干亲本的Wx基因第一内含子+1位碱基类型测定结果表明,与T型亲本相比,G型亲本的直链淀粉含量明显较高,而胶稠度和碱消值没有明显差别。     

转淀粉分支酶反义基因（sbe2b）玉米直链淀粉含量的变异分析

以利用花粉管通道法转化玉米淀粉分支酶基因的反义表达载体的后代中的PCR阳性植株为试材,分析其直链淀粉含量的变异.结果表明:转基因玉米的淀粉分支酶活性有明显的下降,种子的直链淀粉含量比未转化对照有显著提高,T1代种子的直链淀粉含量提高幅度为2.2%～24.9%,最高含量达到了59.4%,表明外源基因的导入有效地抑制了淀粉分支酶基因的表达.转化的淀粉分支酶反义基因能稳定传递,T2代大部分株系的分离比率大致呈3:1,符合孟德尔分离规律;部分株系的分离比率接近于15:1,但卡平方测验不显著.     

转反义蜡质基因纯合株系间直链淀粉含量与RVA谱关系分析

 【目的】排除以往水稻亚种或品种间RVA谱分析时存在着的遗传背景差异,分析转反义蜡质基因不同直链淀粉含量纯合株系间RVA各特征值的差异,更清楚地揭示直链淀粉含量与RVA谱特征值的关系。【方法】采用根癌农杆菌介导共转化和花药培养相结合的方法,培育含有反义蜡质基因的花培纯系,并对各株系进行直链淀粉含量和RVA谱测定和分析,通过比较RVA谱曲线图形及RVA谱特征值的显著性测定,分析相同遗传背景条件下直链淀粉含量对各RVA谱特征值的作用。【结果】在武运粳7号转基因后代中,获得了只含反义蜡质基因、不含潮霉素抗性标记的花培株系77个,经T1H1和T1H2二代直链淀粉含量测定,有19个株系的直链淀粉含量接近对照（对照16.0%）;有50个株系直链淀粉含量比对照下降1%～5%,其中,直链淀粉含量在11.0%～13.9%有30个株系;另外,有8个株系的籽粒为蜡质状,直链淀粉含量降至3.1%～4.0%。不同直链淀粉含量纯系间RVA谱分析发现存在二种RVA谱曲线图形,直链淀粉含量在3.1%～4.0%株系明显不同其它株系,也不同于常规糯稻;对RVA谱曲线图形相同、直链淀粉含量不同的株系进行分类比较,低直链淀粉含量株系的RVA谱曲线最终表现为曲线逐步上升,但没有超越第1个峰值。对不同直链淀粉含量株系类群的各RVA谱特征值进行显著性测定,3.1%～4.0%株系群有5个特征值与其它二个株系群存在极显著差异;11.0%～13.5%株系群有3个特征值与对照16.0%组存在极显著差异;直链淀粉含量与RVA谱各特征值之间优化数学模型显示：峰值时间和消减值与直链淀粉含量关系较为密切。【结论】导入反义蜡质基因,能导致直链淀粉含量降低;通过花药培养能快速获得纯合株系;直链淀粉含量差异不仅影响到各RVA谱特征值,而且还造成RVA谱曲线不同。     

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。     

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.     

杂交水稻育种骨干亲本的米质分析

全面系统地评价杂交水稻骨干育种亲本的米质性状,对于杂交水稻稻米品质性状改良具有重要的指导意义。本研究对274份来源于中国十余个水稻主产区的骨干育种亲本及部分国外来源的育种材料进行了基于类群划分的米质分析。研究结果表明,7项米质性状中,籼稻糙米率、透明度、胶稠度和直链淀粉含量的平均值达到三等以上标准,粳稻糙米率、整精米率、透明度、胶稠度和直链淀粉含量的平均值达到三等以上标准。籼稻米质性状优质达标率变化范围为27.7%~97.7%,粳稻米质性状优质达标率变化范围为16.7%~100%,籼、粳稻垩白性状的优质达标率均较低。籼稻类群中,恢复系和保持系的垩白性状和直链淀粉含量是米质性状改良的重点,常规稻直链淀粉含量较优,热带籼稻中IR43、GIZA 178等米质性状优良,是杂交育种的有利种质资源。筛选出10项米质指标均达三级以上的各类亲本材料共23份,这些育种材料可以作为优异的米质性状改良的基因供体加以利用。另一方面,应加强垩白性状优良的种质资源的引进和利用。     

水稻大粒种质淀粉积累特性研究（摘要）（英文）

[目的]为合理利用水稻大粒种质提供理论依据。[方法]以水稻大粒种质31C122、31C125（千粒重近50 g）和常规水稻品种吉玉粳、赋育333为材料,对其籽粒中糖分和淀粉的积累过程进行对比研究。总糖含量采用苯酚-硫酸法测定,用不同浓度的葡萄糖标准溶液绘制标准曲线。总淀粉含量采用硫酸-蒽酮法测定[10],用不同浓度的葡萄糖标准溶液绘制标准曲线。直链淀粉含量采用农业部颁布的《NY147-88》标准法测定。支链淀粉含量=总淀粉含量-直链淀粉含量。以淀粉积累量W为依变数,开花后天数t（开花日为0）为自变数,应用Logistic方程对淀粉积累过程进行拟合。[结果]供试材料弱势粒总糖含量高于强势粒,且强势粒总糖含量峰值均出现在花后10 d,大粒种质总糖含量低于常规品种;强势粒总淀粉含量高于弱势粒,常规品种总淀粉含量高于大粒种质;供试材料各粒位支链淀粉含量高于直链淀粉,大粒种质直链淀粉含量低于常规品种;大粒种质强势粒总淀粉积累高峰较常规品种强势粒有所推迟,弱势粒总淀粉积累高峰较常规品种有所提前;大粒种质强势粒总淀粉活跃积累期长于常规品种强势粒。[结论]大粒种质的品质好于常规水稻品种。     

隔寒增温超早育秧对水稻产量及质量的影响

为了提高水稻产量与品质,以牡丹江27为试材,研究隔寒增温超早育秧对水稻产量及品质的影响。结果表明,隔寒增温超早育秧水稻的产量和碾米品质优于常规育秧。隔寒增温超早育秧群体有效穗数低于常规育苗,但抽穗期较常规育苗的早5d,每穗粒数、结实率和千粒重均高于常规育苗。稻米灌浆充足成熟度好,稻米整精米率提高8.3%,稻米直链淀粉含量升高,胶稠度较常规育苗略有下降,但蛋白质含量低于常规育苗。     

水稻杂种后代稻米直链淀粉含量的配合力分析

选用稻米直链淀粉含量比较相近的６个粳稻品种,采用Ｇｒｉｆｉｎｇ双列杂交方法四,再结合直链淀粉含量的单粒测定方法,对亲本在Ｆ１、Ｆ２代植株上结实的Ｆ２、Ｆ３稻米直链淀粉含量配合力作了分析。结果表明,稻米直链淀粉含量的ＧＣＡ和ＳＣＡ方差均极显著,表明在本试验中基因加性效应和非加性效应均起重要作用。但,由于二个世代的ｇｃａＭｓ／ｓｃａＭｓ值大于１或显著,且随着世代的推进这一比值又明显增加,因此,稻米直链淀粉含量在杂种后代的表现主要还是由基因加性效应决定。在Ｆ２的胚乳表现中基因非加性效应的作用很大,随着世代的增加,加性基因的作用越显重要。在总的遗传变异中,Ｆ２种子主要以非加性变异为主,Ｆ３种子主要以加性变异为主,但非加性变异仍然占很大比重。配合力效应两世代间相关不显著,亲本稻米直链淀含量的高低与一般配合力的高低并不一致。     

转cry1Ab基因抗虫水稻的培育

采用农杆菌介导的遗传转化法,将cry1Ab基因导入优良三系恢复系明恢63,共获得了92个独立转化株,其中13株为单拷贝。通过发芽试验,获得了11个单拷贝转基因纯合系。ELISA检测结果表明:不同株系其Bt蛋白含量不一样,但杂种的Bt蛋白含量与其亲本一致。室内人工接虫试验及田间抗虫性试验初步表明:5个纯合株系表现高度抗性,且农艺性状与原品种没有显著差异。由此说明,所获得的这些单拷贝转基因株系可作为育种材料,用于抗虫水稻的培育。