CN114149995A - 水稻粒型相关基因drog1及其用途 - Google Patents
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Abstract
本发明公开了调控植物籽粒粒型的基因DROG1及其编码蛋白。调控水稻粒型的基因DROG1的核苷酸序列如SEQ ID No:1所示,其编码的DROG1蛋白的氨基酸序列如SEQ ID No:2所示。本发明还公开了通过提高DROG1表达量来改善水稻粒型的方法,可以用于作物遗传改良。
Description
技术领域
本发明涉及植物基因工程技术领域,涉及一种水稻粒型调控基因DROG1、其编码蛋白DROG1及其用途。
背景技术
水稻是世界上最重要的粮食作物之一,半数以上的人口以水稻为主食,因而对其产量的提高具有迫切的需求。水稻的产量由每株水稻的穗数或有效分蘖数、每穗粒数、粒重和结实率四个主要因素决定,其中粒重又由籽粒大小(体积)和灌浆程度(饱满度)决定[1]。籽粒大小包含粒长、粒宽和粒厚,由于世界上不同地域的顾客对粒型具有不同的偏好性,籽粒大小或形状也是水稻籽粒一个重要的品质性状。
水稻籽粒有禾本科谷类籽粒的典型结构:薄薄的种皮包裹着胚和胚乳,外面包裹着颖壳。储存淀粉和其他营养物质的胚乳占据了成熟种子的大部分,是食物的主要消耗部分。颖壳包含外稃和内稃,不仅提供了保护性的外壳,还形成了灌浆的容器。由于颖壳确定了籽粒的存储容量,限制了籽粒的生长,它在确定籽粒大小上起着主导作用[2]。
颖壳的发育由细胞增殖和细胞扩张协调调节。在发育早期,颖壳中的细胞进行广泛的分裂以增加细胞数量。随后,细胞分裂逐渐减缓,细胞扩张开始,细胞增大。颖壳不同维度上最终的细胞数目和细胞大小决定了籽粒的长度、宽度和厚度,最终影响了细胞大小和形状。近来的研究已经发现了一些数量性状位点(QTL)和调节籽粒大小的关键调节因子。这些调节因子参与了多种信号通路,包含G蛋白信号通路[3-7]、泛素-蛋白酶体通路[8-10]、植物激素信号通路[4,1-13]、MAPK信号通路[14,15]和转录调节因子[16-19]等。
发明内容
本发明的目的在于提供水稻粒型调控基因DROG1,该基因编码植物特有的GRAS家族的一个转录因子成员,主要通过调节细胞分裂方向,改变颖壳横向和纵向的细胞分裂数目,来调控水稻籽粒的长宽比。
本发明还提供了水稻粒型调控基因DROG1的用途,利用该基因对水稻籽粒大小、稻米外观品种和产量的调控,用于作物遗传改良。
具体地,本发明解决其技术问题所采用的技术方案是:
1.调控植物籽粒粒型的基因DROG1,所述基因DROG1的核苷酸序列是如下1)或2):
1)如SEQ ID NO:1所示;
2)与SEQ ID No:1所示的核苷酸序列具有90%以上同源性,且编码相同转录因子功能的蛋白质。
2.调控植物籽粒粒型的蛋白DROG1,所述蛋白DROG1的氨基酸序列是如下1)或2):
1)如SEQ ID NO:2所示,优选地其核苷酸编码序列如SEQ ID NO:1所示;
2)将SEQ ID NO:2的氨基酸序列经过一个或更多个氨基酸残基的取代和/或缺失和/或添加且与粒型相关的由序列1)衍生的氨基酸序列。
3.包含项1所述DNA分子的表达盒、重组载体、转基因细胞系或重组菌。
4.DROG1蛋白在提高植物籽粒产量或调控植物籽粒粒型中的用途,所述DROG1蛋白的氨基酸序列如SEQ ID No:2所示,其中所述籽粒粒型为籽粒的长宽比和/或籽粒的粒重,所述植物为单子叶植物或双子叶植物,优选水稻,更优选日本晴。
5.项4所述的用途,其中所述DROG1蛋白的核苷酸编码序列如SEQ ID No:1所示。
6.项4或5所述的用途,其中所述提高植物籽粒产量通过提高籽粒的长宽比和/或提高籽粒的粒重体现。
7.提高植物籽粒产量的方法,其包括使DROG1蛋白过表达的步骤,其中所述DROG1蛋白的氨基酸序列为SEQ ID No:2,优选地所述DROG1蛋白的核苷酸编码序列如SEQ ID No:1所示,其中所述植物为单子叶植物或双子叶植物,优选水稻,更优选日本晴。
8.项7所述的方法,其中所述提高植物籽粒产量通过提高籽粒的长宽比和/或提高籽粒的粒重体现。
发明人首先在甲基磺酸乙酯诱变的水稻品种日本晴中筛选获得了一个籽粒宽度增加的突变体drog1,将drog1分别与日本晴和籼稻品种明恢63(本实验室)进行杂交,确定drog1突变体受一个单隐性核基因控制并通过图位克隆和转基因互补的方法,对该单隐性核基因进行分子定位及验证,将其命名为DROG1基因。最后用过表达载体,将DROG1基因的序列通过农杆菌转入到野生型日本晴内,发现该基因的表达量升高能够使日本晴籽粒的平均粒长从野生型的7.45毫米增加到8.71毫米,增加了约16.9%,平均千粒重从26.03克增加到28.08克,增加了约7.9%。
附图说明
从下面结合附图的详细描述中,本发明的上述特征和优点将更明显,其中:
图1.野生型与drog1突变体粒型的比较。(A)野生型与drog1突变体的籽粒表型,标尺为1厘米。(B)野生型与drog1突变体的籽粒长度(粒长)、宽度(粒宽)、长宽比和千粒重的比较。
图2.DROG1基因的定位及候选基因确定。
图3.互补载体pCAMBIA1300-DROG1示意图。
图4.DROG1互补植株drog1-com与野生型、drog1突变体的粒型表型。
图5.过表达载体pCAMBIA1300-ubi-DROG1示意图。
图6.野生型与DROG1基因过表达水稻(ubi::DROG1)粒型的比较。(A)野生型与DROG1基因过表达水稻的籽粒表型,标尺为1厘米。(B)野生型与DROG1基因过表达水稻的籽粒长度(粒长)、宽度(粒宽)、长宽比和千粒重的比较。
图7.野生型与drog1突变体、DROG1基因过表达水稻(ubi::DROG1)的颖壳扫描电镜观察。(A)野生型与drog1突变体、DROG1基因过表达水稻的颖壳形态。(B)野生型与drog1突变体、DROG1基因过表达水稻的外稃内侧扫描电镜观察,标尺为100微米。(C)对野生型与drog1突变体、DROG1基因过表达水稻的外稃内侧扫描电镜观察结果的统计分析,包括横向与纵向的外稃大小、细胞大小和细胞数目。
具体实施方式
以下通过实施例来进一步阐明本发明。但是应该理解,所述实施例只是举例说明的目的,并不意欲限制本发明的范围和精神。
本发明中,若非特指,所采用的原料和设备等均可从市场购得或是本领域常用的。下述实施例中的方法,如无特别说明,均为本领域的常规方法。
实施例1.水稻粒型调控基因DROG1的获得
1.drog1突变体的表型观察与遗传分析
粳稻品种日本晴(野生型)经甲基磺酸乙酯诱变的后代中筛选获得的稳定遗传的宽粒突变体,命名为drog1。
图1为野生型与drog1突变体粒型的比较。其中A为野生型与drog1突变体的籽粒表型,B为野生型与drog1突变体的籽粒长度(粒长)、宽度(粒宽)、长宽比和千粒重的比较,结果显示,与野生型相比,drog1的粒长没有明显变化,粒宽和千粒重显著增加,长宽比显著降低。
为了确定drog1的遗传行为,我们将drog1与野生型日本晴进行了正反交试验,F1代都表现为野生型,表明drog1是一个隐性突变体。在F2代分离群体中,野生型单株与突变体单株呈3∶1分离,表明drog1突变受单隐性核基因控制。
表1.drog1与野生型日本晴正反交的F2代分离比的卡方检测
2.水稻粒型调控基因DROG1基因的图位克隆
为了克隆有关drog1的水稻粒型调控基因,命名为DROG1,将drog1突变体与籼稻品种明恢63进行杂交获得F1,利用F1的自交后代构建F2分离群体,选取其中1771株粒型与突变体drog1粒型一样的单株的叶片提取DNA,进行图位克隆。首先利用已有的在日本晴(野生型)与明恢63具有多态性的分子标记对取样群体进行检测,找到与目标性状连锁的分子标记。为了获取与突变基因连锁更加紧密的分子标记,在NCBI(https://www.ncbi.nlm.nih.gov/)中根据定位的区间,将该区间籼稻(9311)和粳稻(日本晴)的序列进行比对,选择具有InDel差异达到至少10bp及以上的位点,用Primier 5.0设计InDel标记,并继续进行多态性检测和取样群体及交换单株的检测,缩小定位区间。用于精细定位的分子标记见表2。
表2用于DROG1定位的分子标记
最终把DROG1基因精确定位在M3和M5两个标记之间,物理距离约为156kb,如图2所示。经过对这一区间内的基因测序,发现了突变体drog1在DROG1基因上存在一段8个碱基的缺失突变。
3.水稻粒型调控基因DROG1基因的功能验证
为了确定是否由于该基因突变导致了drog1突变体的表型,我们通过PCR的方法将野生型日本晴中DROG1基因的全长进行扩增,包含2kb的启动子,全部编码区及1kb的3′UTR,扩增引物为:
F:ggtacccagactcatgatcgagttgc(SEQ ID NO:13)
R:ggtacctggggaacaggaaatttggg(SEQ ID NO:14)
正反向引物均含有KpnI酶切位点,将该片段经酶切回收连接到双元载体pCAMBIA1300(购自Marker Gene公司)中,如图3所示,通过农杆菌侵染的方法转化drog1突变体(转化方法同实施例2),得到转基因植物drog1-com。转基因植株的籽粒都回复到野生型的表型,如图4所示,说明确实是该基因突变导致了drog1突变体的表型,证明本发明正确克隆了水稻粒型调控基因DROG1基因。氨基酸序列分析显示,DROG1是植物特有的GRAS家族的一个转录因子成员。
实施例2.DROG1基因过表达水稻植株的获得
通过PCR的方法扩增从野生型日本晴中克隆DROG1基因的编码序列全长(无内含子),扩增引物为:
F:ggatccatggcgtacatgtgcgcgg(SEQ ID NO:15)
R:aagctttcacctccaagcagacacag(SEQ ID NO:16)
正反向引物分别含有BamHI和HindIII酶切位点,将该片段经酶切连接到然后已连入与ubiquitin启动子和Nos终止子依次酶切连接到的pCAMBIA1300的多克隆位点处,如图5所示。将上述构建好的过表达载体转化到大肠杆菌DH5a感受态细胞(购自北京博迈德基因技术有限公司)中,利用卡那霉素筛选阳性克隆。提取质粒并进行测序鉴定,得到克隆载体中DROG1基因的序列(SEQ ID No:1)完全无误的阳性克隆,然后将该阳性克隆的质粒电击转化至农杆菌EHA105感受态细胞(购自北京博迈德基因技术有限公司)中,进一步利用农杆菌侵染的方法以野生型日本晴为受体进行转基因操作。
转基因操作采用根据2006年Toki等人文章所改进的方法[20],具体如下:
1.预培养
1)去壳的种子(如本实施例中的野生型日本晴种子)经70%乙醇表面消毒1分钟后,经30%NaClO(原液为有效氯10%)消毒30分钟(每50ml滴加1滴Tween20),经无菌水冲洗5次后,重复一次30%NaClO消毒(无需滴加Tween20),并使用无菌水洗5-6次。
2)灭菌后的种子使用无菌的滤纸吸干水分后,铺于N6D培养基(N6D大量,50ml/L;N6D微量,5ml/L;MS有机,5ml/L;Fe-EDTA,5ml/L;2,4-D,2mg/L;CH,0.3g/L;L-proline,2.878g/L;蔗糖,30g/L;Gelrite,4g/L;pH 5.8)上,32℃持续光照培养5-7天,得到愈伤组织。各培养基所用到的母液成分见表3所示。
2.农杆菌侵染及共培养
1)含有相应载体(如本实施例中的DROG1阳性克隆载体)的农杆菌EHA105菌株,在加有25-50mg/L利福平和50mg/L卡那霉素的YEB固体培养基(牛肉膏,5g/L;酵母抽提物,1g/L;胰蛋白胨,5g/L;蔗糖,5g/L;MgSO4·7H2O,0.5g/L;琼脂,15g/L)上28℃暗培养2~3天。挑单菌落于3~5ml加有25-50mg/L利福平和50mg/L卡那霉素的YEB液体培养基中,28℃暗培养约24小时。按1:100接种于50ml AAM培养基(AAM大量,50ml/L;AAM微量,5ml/L;AAM有机,5ml/L;AAM氨基酸,100ml/L;Fe-EDTA,5ml/L;蔗糖,68.5g/L;葡萄糖,36g/L;CH,0.5g/L;Acetosyringone,20mg/L;pH 5.2)中,过夜培养至OD600约0.1。
2)将愈伤组织浸于农杆菌菌液中侵染1.5分钟后,倒去菌液,将愈伤组织置于无菌滤纸上吸去多余菌液并。
3)在含有N6D-As培养基(N6D大量,50ml/L;N6D微量,5ml/L;MS有机,5ml/L;Fe-EDTA,5ml/L;2,4-D,2mg/L;CH,0.3g/L;蔗糖,30g/L;葡萄糖,10g/L;Gelrite,4g/L;Acetosyringone,10-20mg/L;pH5.2)的培养皿上预先铺一层浸有AAM液体培养基的无菌滤纸,并将侵染后的愈伤铺于其上。
4)将培养皿用parafilm封口膜包好置于25℃暗培养3天。
3.筛选及分化
1)共培养后的愈伤组织先用无菌水冲洗2-3次,再用含500mg/L羧苄青霉素的无菌水冲洗2-3次,并用之浸泡30分钟左右(实际可增加清洗次数及浸泡时间)。
2)清洗后的种子使用无菌的滤纸吸干水分后,铺于含50mg/L潮霉素B及400mg/L羧苄青霉素的N6D培养基(N6DS筛选培养基)上,32℃持续光照培养2周后,再转移到新的N6DS培养基上筛选一到两周。
3)只需将生长旺盛的分化愈伤组织转移到含50mg/L潮霉素和250mg/L羧苄青霉素的RE分化培养基(MS大量,50ml/L;MS微量,5ml/L;Fe-EDTA,5ml/L;RE有机,5ml/L;蔗糖,30g/L;Sorbitol,30g/L;CH,2g/L;NAA,0.02mg/L;Kinetin,2mg/L;Gelrite,4g/L;pH 5.8)上,32℃持续光照培养诱导分化3-4周,直至出苗。
4)转移分化出的幼苗至含50mg/L潮霉素和200mg/L羧苄青霉素的HF生根培养基(MS大量,50ml/L;MS微量,5ml/L;Fe-EDTA,5ml/L;RE有机,5ml/L;蔗糖,30g/L;Gelrite,4g/L;pH 5.8)上,继续32℃光照培养。
5)生根后长势良好的转基因幼苗去除培养基,水中培养3天后,移栽至土中。
表3农杆菌侵染法转化水稻相关的培养基母液成分
实施例3.水稻粒型调控基因DROG1过表达植株的表型分析
对DROG1基因过表达植株的籽粒进行观察和比较,如图6所示,A为野生型与DROG1基因过表达水稻(ubi::DROG1)的籽粒表型,B为野生型与DROG1基因过表达水稻的籽粒长度(粒长)、宽度(粒宽)、长宽比和千粒重的比较,结果显示,与野生型相比,DROG1基因过表达植株的粒宽显著降低,粒长、长宽比和千粒重都显著升高。这一结果表明DROG1基因表达量升高可以促进水稻粒长和粒重的增加。
为了确定DROG1基因调控水稻粒型是通过影响细胞大小还是细胞数目,我们对野生型、drog1突变体和DROG1基因过表达植株的外稃内侧细胞进行了扫描电镜观察,并对观察结果进行了统计分析。结果如图7所示,drog1突变体的外稃变宽是由于其横向上细胞数目显著增多,而DROG1基因过表达植株的外稃在纵向上细胞数目的显著增多导致了其外稃增长,提示DROG1基因在颖壳中具有促进细胞纵向分裂、抑制细胞横向分裂的作用,从而通过调节籽粒的长宽比来调控水稻粒型和粒重。
综上所述,本发明利用一个宽粒突变体drog1,通过图位克隆方法克隆了一个粒型调控基因DROG1,该基因编码一个植物特有的GRAS家族转录因子,主要通过调节细胞分裂方向,改变颖壳横向和纵向的细胞分裂数目,来调控水稻籽粒的长宽比
因而,利用本发明提供的DROG1基因的用途——对水稻粒型、稻米外观品种和产量的调控,可以用于作物遗传改良。
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Claims (8)
1.调控植物籽粒粒型的基因DROG1,所述基因DROG1的核苷酸序列是如下1)或2):
1)如SEQ ID NO:1所示;
2)与SEQ ID No:1所示的核苷酸序列具有90%以上同源性,且编码相同转录因子功能的蛋白质。
2.调控植物籽粒粒型的蛋白DROG1,所述蛋白DROG1的氨基酸序列是如下1)或2):
1)如SEQ ID NO:2所示,优选地其核苷酸编码序列如SEQ ID NO:1所示;
2)将SEQ ID NO:2的氨基酸序列经过一个或更多个氨基酸残基的取代和/或缺失和/或添加且与粒型相关的由序列1)衍生的氨基酸序列。
3.包含权利要求1所述DNA分子的表达盒、重组载体、转基因细胞系或重组菌。
4.DROG1蛋白在提高植物籽粒产量或调控植物籽粒粒型中的用途,所述DROG1蛋白的氨基酸序列如SEQ ID No:2所示,其中所述籽粒粒型为籽粒的长宽比和/或籽粒的粒重,所述植物为单子叶植物或双子叶植物,优选水稻,更优选日本晴。
5.权利要求4所述的用途,其中所述DROG1蛋白的核苷酸编码序列如SEQ ID No:1所示。
6.权利要求4或5所述的用途,其中所述提高植物籽粒产量通过提高籽粒的长宽比和/或提高籽粒的粒重体现。
7.提高植物籽粒产量的方法,其包括使DROG1蛋白过表达的步骤,其中所述DROG1蛋白的氨基酸序列为SEQ ID No:2,优选地所述DROG1蛋白的核苷酸编码序列如SEQ ID No:1所示,其中所述植物为单子叶植物或双子叶植物,优选水稻,更优选日本晴。
8.权利要求7所述的方法,其中所述提高植物籽粒产量通过提高籽粒的长宽比和/或提高籽粒的粒重体现。
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