CN116121292A - 一种水稻myb转录因子及其编码蛋白的应用 - Google Patents
一种水稻myb转录因子及其编码蛋白的应用 Download PDFInfo
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Abstract
本发明属于基因工程技术领域,公开了一种水稻MYB类转录因子及其编码蛋白的应用。水稻MYB类转录因子基因OsREPLY的序列号为LOC_Os10g33810(Os10g0478300),可在调控水稻株高、倒伏抗性、花期和产量方面应用。本发明通过系统研究,首次提供了OsREPLY转录因子基因及其编码蛋白的生物学功能。本发明通过CRISPR技术手段获得的osreply突变体材料,导致转基因水稻与野生型对照相比发生株高增加、茎秆变粗抗倒伏、花期改变、产量提高。
Description
技术领域
本发明属于基因工程技术领域,设计一种水稻MYB类转录因子REPLY及其编码蛋白的应用。
背景技术
植物转录因子MYB是近年来发现的一类与调控植物生长发育、生理代谢、细胞的形态和模式建成等生理过程有关的一类转录因子,在植物中普遍存在,同时也是植物中最大的转录家族之一,MYB转录因子在植物的代谢和调控中发挥重要作用。MYB转录因子具有多种生物学功能,广泛参与植物根、茎、叶、花的生长发育,与此同时,MYB基因家族对干旱、盐渍、冷害等非生物胁迫过程也有响应,此外MYB转录因子还与某些经济作物的品质好坏密切相关。
水稻是世界上最重要的粮食作物之一。株高作为重要的农艺性状之一,是水稻株型和产量的核心因素,关系到植株结构、抗倒伏性和产量性能。短茎有利于水稻抗倒伏,但如果植株过短,则会导致生长不足,最终影响水稻的生产潜力。1990年代,矮化育种促成了第一次“绿色革命”。国内外学者对株高与产量的关系进行了研究,提出了理想的株型作为超级稻育种的典范。良好的植物结构保证了作物本身的源-汇-流结构顺畅、合理。高源、大汇、畅流是作物高产的基础。株型改良是水稻育种的主线,株高是株型改良的关键性状。在不改变经济系数的前提下,适当增加株高可以提高叶片的光吸收率,有利于物质的积累,通过提高生物种群的生物产量来提高产量。
本发明从水稻中鉴定了一个MYB转录因子OsREPLY,提出了利用OsREPLY进行水稻株型改良,通过适度增强株高提高叶片光吸收率,促进产量的提高,同时还可以增强植株的抗倒伏能力;此外,该基因3'UTR区的单核苷酸改变可以改变水稻对纬度的适应性,其中单倍型Hap2(CC)可作为更适宜水稻在高纬度地区种植的分子标记。
发明内容
本发明的目的在于提供水稻MYB转录因子基因OsREPLY及其编码蛋白的应用。
本发明的目的通过如下技术方案实现:
水稻MYB类转录因子OsREPLY在增加植物株高、增强抗倒伏性、增强纬度适应性和/或提高产量方面的应用,该基因的MSU编号为LOC_Os10g33810(RAP_Locus编号为:Os10g0478300)。
其中,所述的植物优选单子叶植物,进一步优选水稻、玉米或小麦,特别优选水稻。
作为本发明的一种优选,通过突变、沉默或抑制水稻MYB类转录因子OsREPLY的表达增加植物株高、增强抗倒伏性、增强纬度适应性和/或提高产量。
作为本发明的进一步优选,通过基因编辑手段突变水稻MYB类转录因子OsREPLY的表达增加植物株高、增强抗倒伏性、增强纬度适应性和/或提高产量。
针对所述的水稻MYB类转录因子OsREPLY的基因编辑系统在增加植物株高、增强抗倒伏性、增强纬度适应性或提高产量方面的应用。
其中,所述的植物优选单子叶植物,进一步优选水稻、玉米或小麦,特别优选水稻。
一个水稻MYB类转录因子OsREPLY中的SNP标记,在所述的SNP标记位于REPLY基因3’UTR区域,在水稻基因组物理位置为Chr10-17923788;该位点具有T/C多态性,该位点为TT的为单倍型Hap1,该位点为CC的为单倍型Hap2,光照时间对Hap2的材料的生育期影响更大,具有Hap2单倍型OsREPLY基因的材料更适应高纬度种植;光照时间对Hap2的材料的生育期影响较小,具有Hap1单倍型OsREPLY基因的材料更适应低纬度种植。
本发明所述的SNP标记或其检测试剂在选育纬度适应性水稻品种中的应用,根据该SNP标记的多态性,将水稻材料分为两种单倍型:该位点为TT的为单倍型Hap1,该位点为CC的为单倍型Hap2,光照时间对Hap2的材料的生育期影响更大,具有Hap2单倍型OsREPLY基因的材料更适应高纬度(纬度大于35°)种植;光照时间对Hap2的材料的生育期影响较小,具有Hap1单倍型OsREPLY基因的材料更适应低纬度(纬度小于25°)种植。
本发明所述的SNP标记的检测试剂在鉴定高纬度适应性水稻品种中的应用,根据该SNP标记的多态性,将水稻材料分为两种单倍型:该位点为TT的为单倍型Hap1,该位点为CC的为单倍型Hap2,光照时间对Hap2的材料的生育期影响更大,具有Hap2单倍型OsREPLY基因的材料更适应高纬度种植;光照时间对Hap2的材料的生育期影响较小,具有Hap1单倍型OsREPLY基因的材料更适应低纬度种植。
本发明所述的SNP标记的检测试剂在鉴定低纬度适应性水稻品种中的应用,根据该SNP标记的多态性,将水稻材料分为两种单倍型:该位点为TT的为单倍型Hap1,该位点为CC的为单倍型Hap2,光照时间对Hap2的材料的生育期影响更大,具有Hap2单倍型OsREPLY基因的材料更适应高纬度种植;光照时间对Hap2的材料的生育期影响较小,具有Hap1单倍型OsREPLY基因的材料更适应低纬度种植。
本发明的有益效果:
1、本发明中构建的OsREPLY突变体材料在不同磷浓度土壤中株高显著增加,其高度的增加是由于茎秆节间长度增加造成的(图1)。
2、本发明构建的OsREPLY突变体材料茎秆显著变粗(图2),抗倒伏能力增加,有利于提高水稻在极端环境中的生存能力,保证水稻的产量。
3、本发明构建的OsREPLY突变体材料在不同磷浓度土壤条件下产量均增加(图3),有利于提高水稻在磷营养缺乏条件下的产量。
4、OsREPLY基因的3’UTR的SNP的变化会影响水稻对纬度的适应性(图4),有利于改良水稻品种,培育更高纬度适应性的水稻品种。
附图说明
图1生育后期OsREPLY突变体植株的表型,其株高与野生型植株相比显著增加,茎秆节间长度显著增加。
A在高磷HP和低磷LP土壤中,reply突变后使水稻变高。WT,日本晴野生型;reply,reply突变体材料。
B在高磷HP和低磷LP土壤中,reply突变体与野生型株高统计结果。HP,高磷土壤条件;LP,低磷土壤条件;WT,日本晴野生型材料;reply,reply突变体材料(1-3株系)。
C在高磷HP和低磷LP土壤中,reply突变体和野生型材料主茎茎表型图。WT,日本晴野生型材料;reply,reply突变体材料。
D在高磷HP和低磷LP土壤中,reply突变体和野生型材料主茎穗长和节间表型统计图。
图2生育后期OsREPLY突变体植株抗倒伏表型,极端天气条件下野生型倒伏,突变体不倒伏;突变体材料的茎秆与野生型相比显著变粗,抗倒伏能力增加。A日本晴野生型和reply突变体材料主茎第一个节间横切面表型体视镜照片。WT,日本晴野生型材料;reply,reply突变体材料
B日本晴野生型和reply突变体材料主茎第1、第2、第3节间抗折力统计图。WT,日本晴野生型材料;reply,reply突变体材料;Internode I、II、III分别表示节间1、节间2、节间3.
C高磷土壤中,日本晴野生型WT与reply突变体抗倒伏表型照片。
图3生育后期OsREPLY突变体植株产量统计,与野生型植株相比显著增加。A在高磷HP和低磷LP土壤中WT和reply突变体材料单株产量表型图。HP,高磷土壤条件;LP,低磷土壤条件;WT,日本晴野生型材料;reply,reply突变体材料。
B在高磷HP和低磷LP土壤中WT和reply突变体材料单株产量数据统计图。
图4OsREPLY不同单倍型花期比较及不同品种中单倍型分析。
A在长、短日照条件下,日本晴背景和武运粳背景条件下,野生型与REPLY超表达材料花期表型图。Nip-WT,日本晴野生型;Nip-Ox,日本晴背景REPLY超表达;WYJ-WT,武运粳野生型;WYJ-Ox,武运粳背景REPLY超表达。LD,长日照条件(南京);SD,短日照条件(海南)。
B日本晴背景野生型和武运粳背景野生型在长、短日照条件下花期变化差值比较统计图。武运粳背景野生型花期变化受长、短日照条件影响较大。
C REPLY的两种单倍型(Hap1——代表材料为日本晴;Hap2——代表材料为武运粳)在不同品种中的分布情况。发现受光照条件影响较大的Hap2在高纬度分布比例较高,受光照条件影响较小的Hap1在高纬度分布比例较低。Hap1(代表材料为日本晴);Hap2(代表材料为武运粳)。
图5表达载体质粒pH-Ubi-Cas9-7和pOs-sgRNA质粒图谱
图6表达载体质粒pCAMBIA1305-GUSPlus图谱
具体实施方式
实施例1OsREPLY基因敲除载体构建及工程菌构建
基因编辑技术飞速发展,CRISPR/Cas9系统作为一种新兴的基因定点突变编辑技术正在逐步成熟并且已经成功应用于多种动植物物种,促进了基因功能的研究。应用CRISPR/Cas9技术可以直接在基因组水平对目的基因的特定DNA序列进行编辑,造成碱基对缺失、增加或者突变,从而导致蛋白移码突变或蛋白翻译提前终止等结果,达到敲除目的基因的目的。
利用Miao等(2013)发表的文章附录文件中所提供的水稻全基因组的基因Spacer数据库(http://cbi.hzau.edu.cn/cgi-bin/CRISPR),输入OsREPLY基因登录号LOC_Os10g33810,搜索OsREPLY的基因特异的Spacers。我们从搜索到的Spacers中挑选了3条,挑选Spacers时则应注意:1)挑选不带星号的Spacer;2)Spacer的GC含量高为宜;3)Spacer的特异性(近源物种间一个家族基因的序列相似性比对,Spacer靠后12个bp比较重要,12bp+NGG应重点在NCBI网站上进行BLAST,确保选择的Spacer不会脱靶);4)Spacer相邻的3’序列必须为NGG。将最终确定的Spacer两条互补链的5’端加上BsaI酶切位点,设计引物(表1)。将互补的两条单链引物在PCR仪上进行退火,合成双链,反应条件:95℃30s,72℃2min,65℃2min,45℃2min,37℃2min,25℃2min,10℃hold。将退火合成的双链DNA片段和经BsaI酶切的pOs-sgRNA载体片段用T4 DNA ligase酶连过夜,转化DH5α大肠感受态,测序正确后保存代用。
将含有OsREPLY基因的Spacer的pOs-sgRNA质粒和表达载体质粒pH-Ubi-Cas9-7(图5)在LR Clonase II enzyme的作用下进行Gateway LR反应,产物转化DH5α大肠感受态,获得重组质粒,质粒经测序验证无误后,2500V电击转化EHA105农杆菌感受态中,-70℃保存备用。
表1OsREPLY基因特异spacers设计
实施例2OsREPLY超表达材料载体构建及工程菌构建
针对基因过量表达载体的构建,利用DNAMAN软件对OsREPLY编码区序列进行限制性酶切位点分析,设计含有相应酶切位点的PCR扩增引物,引物序列为:REPLY-cDNA-F:5'-TTGGATCCATGGGGAGGGCGCCGTGCTGC-3’;REPLY-cDNA-R:5’-TTAAGCTTTTACAAGACGGCCAAATCCCT-3’。以含有OsREPLY全长cDNA的克隆载体为模板,进行PCR扩增。PCR产物经凝胶回收纯化后克隆到pEASY-Blunt克隆载体上,阳性克隆经酶切验证并测序正确后,经BamHI和HindIII双酶切后,导入pCAMBIA1305-GUSPlus载体(图6)中,获得的重组质粒经酶切验证及测序后,待用。
实施例3农杆菌介导的水稻转基因
将含有不同表达载体质粒的农杆菌侵染水稻(Nipponbare)成熟胚愈伤组织,(根据实验需要选择不同背景野生型的愈伤组织。本实验中需要侵染日本晴和武运粳两种背景材料的愈伤组织)。暗条件共培养2.5天,转入含有50mg/L潮霉素和500mg/L羧苄青霉素的培养基上进行第一轮筛选;3周后移至含有80mg/L潮霉素和500mg/L羧苄青霉素的培养基上进行第二轮筛选;2周后,将新长出的抗性愈伤转入分化培养基中进行分化,一个月后出苗。将分化出的幼苗转移至生根培养基中,根生7天,经过炼苗后获得完整的转基因水稻植株。
实施例4转基因osreply突变体植株鉴定
转基因材料阳性苗鉴定:剪取一小片T0代转基因苗叶片,用微量DNA提取法提取每个单株的基因组DNA,设计Cas9基因特异性引物,以提取的DNA为模板,进行PCR扩增,核酸凝胶电泳检测。Cas9基因检测引物序列:Cas9-F:5'-ACAAGGGCAGGGATTTCG-3',Cas9-R:5'-ACTGGTGGATGAGGGTG GC-3',产物大小为827bp,若扩增出相应大小的DNA片段则为转基因阳性苗。
OsREPLY基因突变位点鉴定:设计OsREPLY基因片段特异性引物(扩增片段范围涵盖三个Spacers),以上述所提的DNA为模板,进行PCR扩增,核酸凝胶电泳检测。OsREPLY基因片段特异扩增引物序列为:REPLY-CRISPR-F:5’-TAAATACGCGCCACCCCGCC-3’;REPLY-CRISPR-R:5’-AGGCCATAAGGTGATGGGACAGT-3’,产物大小760bp。胶回收扩增出的DNA片段,克隆至pEasy-Blunt克隆载体,转化DH5α,每个转化至少送三个阳性克隆至公司测序,分析测序结果,挑选出OsREPLY基因发生有效突变的转基因植株,扩繁收获T1代种子进行后续实验。
实施例5转基因REPLY-Ox超表达材料的鉴定
对获得的REPLY-Ox过量表达植株通过GUS报告基因的表达来鉴定阳性苗,并通过RT-qPCR方法进行超表达效果鉴定,定量引物序列如表2所示。
表2OsREPLY基因RT-qPCR引物列表
实施例6OsREPLY突变体材料成熟期表型统计
将OsREPLY突变体和日本晴野生型材料浸种一天后进行催芽,种子露白出芽后于田间育秧,当幼苗生长至一定程度(30天左右)即可移栽并进行小区处理。每个品种在不同处理条件下各设置三个重复小区,每个小区以11株×12株种植,行距20cm,株距20cm,于分蘖期、扬花和成熟期三个时期进行采样分析。
去除小区最外两层,选取10株水稻统计株高及分蘖数,株高以水稻和土地的接触面为底部、形态学顶端为最高点测量,并对成熟期各单株进行单株产量的称重,统计株高、节间长度及产量等数据。
不管在低磷还是高磷土壤条件下,reply突变体材料的株高较野生型相比株高均显著增高。说明REPLY基因负调控水稻的株高,作为一个负调控株高的关键转录因子起作用(图1)。我们观察到reply突变体材料虽然株高增加,但是由于其茎秆变粗壮,使得reply突变体材料抗倒伏能力增强(图2),结合统计其单株产量的数据,我们发现REPLY突变后,株高增加,抗倒伏能力增加并且可以提高产量(图3),可以作为非常优良的分子标记对水稻品种进行改良。
实施例7OsREPLY单倍型分析及表型关联
REPLY基因在不同水稻品种中具有多个SNP变异。我们利用3k数据库(http://www.rmbreeding.cn/index.php)对REPLY基因进行分析,发现在REPLY基因3’UTR区域的一个SNP位点(该SNP位点在水稻基因组物理位置为Chr10-17923788)对水稻纬度的适应性关联密切。根据该位点的SNP可以将水稻分成Hap1(TT)和Hap2(CC)两种单倍型材料。Hap1 3’UTR序列如SEQ IDNO.1所示,SNP位点位于第194bp,Hap1在该位点基因型为T;Hap2 3’UTR序列序列如SEQ ID NO.2所示,SNP位点位于第194bp,Hap1在该位点基因型为C。Hap1典型代表为日本晴,Hap2典型代表为武运粳7号。
将日本晴和武运粳背景的OsREPLY超表达材料和野生型种植在南京农业大学白马教学基地(江苏南京)和南京农业大学南繁基地(海南乐东),统计花期并探究花期差异的原因。在对日本晴和武运粳两个背景的野生型和REPLY超表达材料进行花期统计中,我们发现,由于两个野生型对于长短日照响应的能力不同,使得在LD条件(南京)和SD条件下(海南)两背景条件下的REPLY的超表达的开花时间较野生型有完全相反的表型。对这两个典型的单倍型材料长短日照条件下的开花时间进行分析后,我们可以得到光照时间对Hap2的材料的生育期影响更大,对Hap1的材料的生育期影响较小(图4A,B)。
分析日本晴和武运粳序列的差异发现可能造成该差异的原因,再利用3k数据库(https://www.rmbreeding.cn/index.php)分析OsREPLY的单倍型,根据该位点SNP的变化可以将水稻分成Hap1(代表材料为日本晴)和Hap2(代表材料为武运粳),将不同品种的起源地理位置与单倍型关联后进行画图(图4C),从图中可以看出,随着纬度的增加,Hap2所占比例逐渐增加,Hap1所占比例逐渐降低,由此可得出REPLY基因的3’UTR区域的单碱基改变可以影响水稻对纬度的适应性,Hap2类型的REPLY更适宜作为选育适应高纬度品种的分子标记,Hap1类型的REPLY更适宜作为适应低纬度品种的分子标记。
Claims (10)
1.水稻MYB类转录因子OsREPLY在增加植物株高、增强抗倒伏性、增强纬度适应性和/或提高产量方面的应用,该基因的MSU编号为LOC_Os10g33810。
2.根据权利要求1所述的应用,其特征在于所述的植物为单子叶植物,优选自水稻、玉米或小麦中的任意一种。
3.根据权利要求2所述的应用,其特征在于通过突变、沉默或抑制水稻MYB类转录因子OsREPLY的表达增加植物株高、增强抗倒伏性、增强纬度适应性和/或提高产量。
4.根据权利要求3所述的应用,其特征在于通过基因编辑手段突变水稻MYB类转录因子OsREPLY的表达增加植物株高、增强抗倒伏性、增强纬度适应性和/或提高产量。
5.针对权利要求1所述的水稻MYB类转录因子OsREPLY的基因编辑系统在增加植物株高、增强抗倒伏性、增强纬度适应性和/或提高产量方面的应用。
6.根据权利要求5所述的应用,其特征在于所述的植物为单子叶植物,优选自水稻、玉米或小麦中的任意一种。
7.一个水稻MYB类转录因子OsREPLY中的SNP标记,其特征在于在所述的SNP标记位于REPLY基因3’UTR区域,在水稻基因组物理位置为Chr10-17923788;该位点具有T/C多态性,该位点为TT的为单倍型Hap1,该位点为CC的为单倍型Hap2,光照时间对Hap2的材料的生育期影响更大,具有Hap2单倍型OsREPLY基因的材料更适应高纬度种植;光照时间对Hap2的材料的生育期影响较小,具有Hap1单倍型OsREPLY基因的材料更适应低纬度种植。
8.权利要求7所述的SNP标记或其检测试剂在选育纬度适应性水稻品种中的应用,其特征在于根据该SNP标记的多态性,将水稻材料分为两种单倍型:该位点为TT的为单倍型Hap1,该位点为CC的为单倍型Hap2,光照时间对Hap2的材料的生育期影响更大,具有Hap2单倍型OsREPLY基因的材料更适应高纬度种植;光照时间对Hap2的材料的生育期影响较小,具有Hap1单倍型OsREPLY基因的材料更适应低纬度种植;所述的高纬度为纬度大于35°,所述的低纬度为纬度小于25°。
9.权利要求7所述的SNP标记的检测试剂在鉴定高纬度适应性水稻品种中的应用,其特征在于根据该SNP标记的多态性,将水稻材料分为两种单倍型:该位点为TT的为单倍型Hap1,该位点为CC的为单倍型Hap2,光照时间对Hap2的材料的生育期影响更大,具有Hap2单倍型OsREPLY基因的材料更适应高纬度种植;光照时间对Hap2的材料的生育期影响较小,具有Hap1单倍型OsREPLY基因的材料更适应低纬度种植。
10.权利要求7所述的SNP标记的检测试剂在鉴定低纬度适应性水稻品种中的应用,其特征在于根据该SNP标记的多态性,将水稻材料分为两种单倍型:该位点为TT的为单倍型Hap1,该位点为CC的为单倍型Hap2,光照时间对Hap2的材料的生育期影响更大,具有Hap2单倍型OsREPLY基因的材料更适应高纬度种植;光照时间对Hap2的材料的生育期影响较小,具有Hap1单倍型OsREPLY基因的材料更适应低纬度种植。
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CN118028516A (zh) * | 2023-06-14 | 2024-05-14 | 中国热带农业科学院热带生物技术研究所 | 一种龙眼红色果皮表型的筛选标记 |
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