CN112979774B - 水稻OsAQP基因在改变水稻粒形中的应用 - Google Patents
水稻OsAQP基因在改变水稻粒形中的应用 Download PDFInfo
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- CN112979774B CN112979774B CN202110004928.7A CN202110004928A CN112979774B CN 112979774 B CN112979774 B CN 112979774B CN 202110004928 A CN202110004928 A CN 202110004928A CN 112979774 B CN112979774 B CN 112979774B
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Abstract
本发明公开了水稻OsAQP基因在改变水稻粒形中的应用，属于农业和分子生物学技术领域。本发明所述水稻OsAQP基因序列如SEQ ID NO.1所示，大小为961bp。本发明将水稻水通道蛋白编码基因OsAQP构建过表达植物载体，通过农杆菌介导的方法转化水稻愈伤组织获得T3代过表达OsAQP基因的转基因水稻，与对照相比，转基因水稻的粒长减小7.99％‑9.57％、粒宽减小5.04％‑6.43％、粒厚减小7.05％‑8.78％。
Description
技术领域
本发明属于农业和分子生物学技术领域，具体涉及水稻OsAQP基因在改变水稻粒形中的应用。
背景技术
水稻（Oryza sativa L.）是世界上最重要的粮食作物之一，为全球一半以上的人口提供食物和营养来源，是保障粮食安全和实现农业可持续发展的主粮作物[1]。
生产上，水稻的单株有效穗数、每穗实粒数和粒重等因素决定了产量，而水稻粒重又受谷粒粒形、大小及成熟度等因素影响。根据籽粒的三维结构将粒形构成划分为粒长、粒宽和粒厚[2]。因此，粒形是决定水稻产量的重要农艺性状之一。粒形及籽粒大小不仅直接影响水稻的产量，还与水稻的品质有着密切的关系，因此作为水稻品种的标志性特征之一，特定的粒形对培育筛选高产优质水稻具有非常重要的意义。
水稻粒形是典型的数量性状，粒长、粒宽、长宽比一般受多基因控制[3]。随着水稻基因组计划的完成，关于水稻粒形的研究取得了较大的进展，目前已经定位了400多个与粒形相关的QTLs，并已克隆了一批控制水稻粒形的基因[4-6]，例如GS3、GW2、GW5、qSW5、GW8、GS5和HGW等[7-10]，这些基因分别影响水稻谷粒的粒长、粒宽与粒厚，并使谷粒粒形发生变化。
参考文献：
[1]郭韬,余泓,邱杰等.中国水稻遗传学研究进展与分子设计育种[J].中国科学:生命科学,2019,49(10):1185-1212。
[2]高志强,占小登,梁永书等.水稻粒形性状的遗传及相关基因定位与克隆研究进展[J].遗传,2011,33(04):314-321。
[3]朱业宝,郭玉春,梁康迳等.水稻粒形调控基因的研究进展[J].福建农林大学学报:自然科学版, 2015,44(1):1-7。
[4]Huang R, Jiang L, Zheng J, et al. Genetic bases of rice grainshape: so many genes, so little known[J]. Trends in Plant Science, 2013, 18(4) :218。
[5]Zang Q, Wing R A. Genetics and Genomics of Rice[M]. New York:Springer, 2013:237。
[6]Fan C, Xing Y, Mao H, et al. GS3,a major QTL for grain length andweight and minor QTL for grain width and thickness in rice, encodes aputative transmembrane protein[J]. Theor Appl Genet,2006, 112:1164-1171。
[7]Song X J, Huang W, Shi M, et al. A QTL for rice grain width andweight encodes a previously unknown RING-type E3 ubiquitin ligase[J]. NatGenet, 2007, 39:623-630。
[8]Weng J, Gu S, Wan X, et al. Isolation and initial characterizationof GW5,a major QTL associated with rice grain width and weight[J]. Cell Res,2008, 18:1199-1209。
[9]Zhang X, Wang J, Huang J, et al. Rare allele of OsPPKL1 associatedwith grain length causes extra-large grain and a significant yield increasein rice[J]. Proc Natl Acad Sci USA,2012, 109:21534-21539。
[10]Zhang X, Wang J, Huang J, et al. Rare allele of OsPPKL associatedwith grain length causes extra-large grain and a significant yield increasein rice[J]. Proceedings of the National Academy of Sciences, 2012, 109 (52) :21534-21539。
发明内容
本发明的目的是提供了水稻OsAQP基因在改变水稻粒形中的应用，即将含有OsAQP基因的表达载体转染到水稻中，然后培育水稻获得T3代过表达OsAQP基因的转基因水稻。
本发明为实现上述目的采用如下技术方案，水稻OsAQP基因在改变水稻粒形中的应用，其特征在于：所述水稻OsAQP基因序列如SEQ ID NO.1所示，大小为961bp。
本发明所述的水稻OsAQP基因在改变水稻粒形中的应用，其特征在于具体过程为：设计引物P1：5’-AACTGCAGATGCCGATCCGCAACATC-3’，含有PstI酶切位点和引物P2：5’-AACCTAGGGTAGTCGGTGGTGGGGA-3’，含有AvrII酶切位点，通过PCR扩增为OsAQP基因cDNA编码区5’端和3’端分别添加PstI和AvrII酶切位点，连接到植物表达载体pCAMBIA1302的相应位点，再通过农杆菌介导的方法转化水稻愈伤组织获得T3代过表达OsAQP基因的转基因水稻。
本发明与现有技术相比具有以下有益效果：本发明将水稻水通道蛋白编码基因OsAQP构建过表达植物载体，通过农杆菌介导的方法转化水稻愈伤组织获得T3代过表达OsAQP基因的转基因水稻，与对照相比，转基因水稻的粒长减小7.99%-9.57%、粒宽减小5.04%-6.43%、粒厚减小7.05%-8.78%。
附图说明
图1为对照水稻与OsAQP基因过表达转基因水稻的种子表型对比，WT：野生型水稻，#11-2，#10-3，#9-2： OE-OsAQP转基因水稻。
图2为对照水稻和OsAQP基因过表达转基因水稻T3代不同株系水稻粒长的统计学比较，WT：野生型水稻，#11-2，#10-3，#9-2： OE-OsAQP转基因水稻；（n=8，*P<0.05，**P<0.01）。
图3为对照水稻和OsAQP基因过表达转基因水稻T3代不同株系水稻粒宽的统计学比较，WT：野生型水稻，#11-2，#10-3，#9-2：OE-OsAQP转基因水稻；（n=8，*P<0.05，**P<0.01）。
图4为对照水稻和OsAQP基因过表达转基因水稻T3代不同株系水稻粒厚的统计学比较，WT：野生型水稻，#11-2，#10-3，#9-2：OE-OsAQP转基因水稻；（n=8，*P<0.05，**P<0.01）。
图5为OsAQP过表达转基因水稻T3代的PCR检测，通过PCR检测可以筛选到阳性植株，条带正确且亮的代表阳性植株，1：DNA Marker DL2000；2：以水为模板的阴性对照；3：WT；4-11：OE-OsAQP转基因水稻植株。
图6为OsAQP过表达转基因水稻T3代的表达量检测，通过表达量检测可以检测该基因在此植株中的表达水平，从图中我们可以得出转基因水稻中该基因的表达量都有了不同程度的提高，WT：野生型水稻；1-8：OE-OsAQP转基因水稻T3代植株。
具体实施方式
以下通过实施例对本发明的上述内容做进一步详细说明，但不应该将此理解为本发明上述主题的范围仅限于以下的实施例，凡基于本发明上述内容实现的技术均属于本发明的范围。
实施例1
1、水稻OsAQP基因过表达载体的构建
设计引物P1：（5’-AACTGCAGATGCCGATCCGCAACATC-3’，含有PstI酶切位点）和引物P2：（5’-AACCTAGGGTAGTCGGTGGTGGGGA-3’，含有AvrII酶切位点），通过PCR扩增OsAQP基因cDNA编码区，并为其5’端和3’端分别添加PstI和AvrII酶切位点，连接到植物表达载体pCAMBIA1302（购自CAMBIA公司）的相应位点。连接产物转化大肠杆菌DH5α，在含有卡那霉素的LB平板上筛选阳性转化子，经酶切和PCR鉴定后，测序确认序列未发生突变。
2、水稻的遗传转化、筛选和T3代转OsAQP基因水稻性状的分析
采用常规的水稻遗传转化方法，利用农杆菌浸染法将OsAQP基因过表达载体转化至水稻愈伤组织（将去颖壳的水稻种子点播在愈伤诱导培养基N6D2上，使水稻种子的胚露出培养基。培养7天后，水稻黄色胚性愈伤形成。除去芽和根后，将愈伤置于新的的N6D2培养基上继续诱导，大约10天能够形成比较坚硬的愈伤组织，可用于农杆菌侵染），利用潮霉素筛选阳性愈伤组织，进一步诱导培育再生阳性转基因植株，并进行分子鉴定。转基因水稻筛选至T3代时，对同一批次收获的成熟对照水稻和T3代转基因水稻进行观察，分别测量其谷粒的粒长、粒宽和粒厚，进行统计学分析，统计样本各8株，每个株系重复3次，统计结果显示未转基因的对照水稻的平均粒长为0.7335±0.0152，平均粒宽为0.3257±0.0096，平均粒厚为0.2257±0.0014；而转基因水稻的平均粒长为0.6745±0.0251，平均粒宽为0.3073±0.0168，平均粒厚为0.2083±0.0023，与对照相比，转基因水稻的粒长减小7.99%-9.57%、粒宽减小5.04%-6.43%、粒厚减小7.05%-8.78%。
以上实施例描述了本发明的基本原理、主要特征及优点，本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明原理的范围下，本发明还会有各种变化和改进，这些变化和改进均落入本发明保护的范围内。
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Claims (2)
1.水稻OsAQP基因在改变水稻粒形中的应用，其特征在于：所述水稻OsAQP基因序列如SEQ ID NO.1所示，大小为961bp。
2.根据权利要求1所述的水稻OsAQP基因在改变水稻粒形中的应用，其特征在于具体过程为：设计引物P1：5’-AACTGCAGATGCCGATCCGCAACATC-3’，含有PstI酶切位点， 和引物P2：5’-AACCTAGGGTAGTCGGTGGTGGGGA-3’，含有AvrII酶切位点，通过PCR扩增为OsAQP基因cDNA编码区5’端和3’端分别添加PstI和AvrII酶切位点，连接到植物表达载体pCAMBIA1302的相应位点，再通过农杆菌介导的方法转化水稻愈伤组织获得T3代过表达OsAQP基因的转基因水稻。
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