CN112126710B - 水稻源抗虫相关基因OsLRR6及其编码产物与应用 - Google Patents
水稻源抗虫相关基因OsLRR6及其编码产物与应用 Download PDFInfo
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Abstract
本发明公开了一种水稻源抗虫相关基因OsLRR6及其编码产物与应用,该抗虫基因为SEQ ID No.1的DNA序列。该基因完整编码框为SEQ ID No.1中的第42到第794的碱基序列,编码250个氨基酸残基的小分子量蛋白,见SEQ ID No.2。研究发现该基因全长DNA序列SEQ ID No.1中的第200到第219的DNA片段与水稻的抗虫性密切相关,利用CRISPR/Cas9技术突变该DNA片段、降低该基因的表达水平能增强水稻对稻飞虱的抗性。本发明将在作物育种,特别是在水稻抗虫育种中得到广泛应用。
Description
技术领域
本发明属于生物技术领域,尤其涉及一种水稻源抗虫相关基因OsLRR6及其编码产物与应用。
背景技术
水稻(Oryza sativa L.)是世界上最重要的粮食作物之一,其安全生产满足了全球数十亿人的生活所需。稻飞虱,包括褐飞虱(Nilaparvata lugens)、白背飞虱(Sogatellafurcifera)、灰飞虱(Laodelphax striatellus),是水稻最具破坏性的害虫,对水稻生产构成重大威胁。它们定居在水稻植株茎基部,吮吸韧皮部汁液并产卵,对水稻植株造成直接伤害,严重发生会导致水稻植株死亡,这种情况通常被称为“虱烧”。此外,这三种稻飞虱也是水稻主要病毒病的传播媒介。面对日益严峻的人口增长、用药成本增加、有毒有害物质残留等环境污染以及害虫再猖獗等问题,使得研究培育具有安全、稳产、增产、抗虫等性状的水稻新品种迫在眉睫。
寄主植物抗性的提高已被公认是防治稻飞虱最实用、经济、环保的策略之一。令人注意的是,迄今为止,研究人员在水稻抗稻飞虱主要基因/数量性状位点(QTLs)的鉴定和定位研究方面取得了重大进展,已经从水稻中分别鉴定出至少30个、14个和34个抗褐飞虱、白背飞虱和灰飞虱的主要基因/QTLs。然而,由单一抗性基因控制的改良品种很可能并不具备对多种或新进化的生物型稻飞虱产生抗性的能力。近几十年来的报道表明,这三种飞虱开始在东亚某一种植季节同时出现,甚至在中国某些地区的同一田间同时出现。因此,鉴定更有效、更广谱的抗稻飞虱的水稻种质、发现新的广谱性抗稻飞虱基因并将其整合到水稻品种中去,是水稻生产中的巨大挑战,同样也对实现稻飞虱可持续控制具有十分重要的意义。
发明内容
本发明的目的在于针对现有技术的不足,提供一种水稻源抗虫相关基因OsLRR6及其编码产物与应用。
本发明的目的是通过以下技术方案来实现的:一种水稻源抗虫相关基因OsLRR6,它具有SEQ ID No.1的DNA序列。
一种上述水稻源抗虫相关基因OsLRR6编码的蛋白,它是包含SEQ ID No.2的氨基酸序列的蛋白质,或者是将SEQ ID No.2的氨基酸残基经过一个或多个氨基酸残基的取代、缺失或者添加且具有与SEQ ID No.2的氨基酸残基序列相同活性的由SEQ ID No.2衍生的蛋白质。
一种上述水稻源抗虫相关基因OsLRR6的DNA片段,该DNA片段为基因OsLRR6全长DNA序列SEQ ID No.1中的第200到第219的碱基序列。
一种上述水稻源抗虫相关基因OsLRR6的DNA片段在转基因水稻中的应用。
一种上述水稻源抗虫相关基因OsLRR6的DNA片段在抗虫水稻育种中的应用。
本发明的有益效果是:利用抑制消减杂交(SSH)和反转录PCR(RT-PCR)技术分离并克隆了OsLRR6基因;利用荧光定量PCR(qRT-PCR)方法分析了OsLRR6基因在褐飞虱产卵雌成虫为害前后的表达情况;利用CRISPR/Cas9基因编辑技术和农杆菌介导的植物转化技术获得了OsLRR6基因突变的转基因植株,并发现了基因OsLRR6的DNA片段在水稻对稻飞虱的抗性中发挥着十分重要的作用。该基因的分离克隆及其基因片段的应用,对作物的抗虫育种,尤其是抗稻飞虱的水稻品种的培育,具有十分重要的指导和促进作用。
附图说明
图1是OsLRR6全长基因PCR扩增产物凝胶电泳图;图中,M为DNA标准分子量;1泳道为OsLRR6基因PCR电泳结果;
图2是褐飞虱产卵雌成虫为害前后OsLRR6基因的表达情况(均值+标准误,n=5)。对照:套入玻璃罩但未被褐飞虱产卵雌成虫为害的常规水稻植株叶鞘;褐飞虱为害:在玻璃罩内接入褐飞虱产卵雌成虫为害的常规水稻植株叶鞘。星号表示处理组与对照组存在显著差异(*:P<0.05;**:P<0.01;Student’s t-test);
图3是用于突变OsLRR6基因的CRISPR/Cas9载体构建示意图;
图4是OsLRR6在转基因突变体中的沉默效果图(均值+标准误,n=5)。褐飞虱产卵雌成虫为害不同时间,常规植株(WT)、突变品系(K1和K3)中OsLRR6基因的表达量。a、b等不同字母表示对照组与处理组存在显著差异(P<0.05,Duncan’s multiple-range test);
图5是突变OsLRR6后降低了褐飞虱产卵雌成虫取食和产卵的偏好性(均值+标准误,n=10)。(A-B)表示褐飞虱产卵雌成虫在常规植株(WT)、突变植株(K1或K3)上的取食头数和产卵率。星号表示处理组与对照组存在显著差异(*:P<0.05;**:P<0.01;Student’s t-test);
图6是突变OsLRR6后降低了褐飞虱卵的孵化率(均值+标准误,n=10)。常规植株(WT)、突变品系(K1和K3)上褐飞虱卵的孵化率。a、b等不同字母表示对照组与处理组存在显著差异(P<0.05,Duncan’s multiple-range test);
图7是突变OsLRR6后增强了水稻对褐飞虱产卵雌成虫的耐受性(n=10)。25头褐飞虱产卵雌成虫为害13天后,常规植株(WT)与突变品系(K1和K3)的耐害表型;
图8是突变OsLRR6后降低了田间褐飞虱和白背飞虱的种群密度(均值+标准误,n=3)。A-B表示每丛常规水稻植株(WT)、突变品系(K1和K3)上褐飞虱成虫和若虫数量;C-D表示每丛常规水稻植株(WT)、突变品系(K1和K3)上白背飞虱的成虫和若虫数量。a、b等不同字母表示对照组与处理组存在显著差异(P<0.05,Duncan’s multiple-range test)。
具体实施方式
本发明利用SSH、RT-PCR和RACE技术,获得OsLRR6的全长序列。首先从SSH克隆库中分析获得OsLRR6基因片段,以此片段设计正向和反向引物,分别进行3’-RACE和5’-RACE获得该基因的5’端和3’端基因PCR片段,并测序拼接获得DNA序列。在拼接DNA序列的5’端和3’端非编码区设计引物OsLRR6-F1和OsLRR6-R1。提取褐飞虱为害24h的水稻叶鞘的总RNA并反转录成cDNA。以cDNA为模板、OsLRR6-F1和OsLRR6-R1为引物进行PCR反应,即得到OsLRR6全长序列并测序验证。其次,利用qRT-PCR研究OsLRR6的表达情况,结果表明褐飞虱产卵雌成虫危害诱导了OsLRR6的转录。再次,设计靶点序列并利用CRISPR/Cas9技术和农杆菌转化法得到OsLRR6突变的转基因植株,通过生物学测定证明OsLRR6基因突变后能够显著增加水稻对褐飞虱的抗性。该基因片段的分离和克隆以及生物学功能的分析,对于作物的抗虫育种,尤其对水稻抗褐飞虱育种将起到重要的促进作用。
实现本发明的具体技术步骤如下:
1、水稻OsLRR6基因的分离和序列分析
首先从SSH克隆库中分析获得OsLRR6基因片段,以此片段设计正向引物和反向引物,分别进行3’-RACE和5’-RACE获得该基因的5’端和3’端基因PCR片段,并测序拼接获得DNA序列。根据拼接的DNA序列,我们在其5’端和3’端非编码区设计引物OsLRR6-F1和OsLRR6-R1,以褐飞虱为害24h的水稻叶鞘总RNA反转录的cDNA为模板,使用KOD FX高保真酶(TOYOBO)进行PCR反应,获得OsLRR6基因全长序列。OsLRR6序列见SEQ ID No.1。根据该序列的开放阅读框(ORF),推算出该基因编码蛋白的氨基酸序列,见SEQ ID No.2。
2、褐飞虱为害前后的OsLRR6表达特征分析
水稻经褐飞虱产卵雌成虫为害0、0.5、1、3、8、12、24、48h后,取为害部位叶鞘。利用MiniBEST Plant RNA Extraction Kit(TaKaRa)试剂盒提取总RNA,并利用超微量蛋白核酸分光光度计(BioDrop)和甲醛凝胶变性电泳检测与评估所得RNA的浓度、纯度与质量。用PrimeScriptTMRT Master Mix(TaKaRa)试剂盒将500ng总RNA反转录成cDNA,具体操作参照产品说明书。
荧光定量PCR(qRT-PCR)检测使用Premix Ex Taq[Probe qPCR](TaKaRa)酶预混液配制反应体系,使用CFX96TMReal-Time system(Bio-RAD)定量PCR仪检测荧光信号。以水稻OsACTIN基因(TIGR ID:LOC_Os03g50885)作为内参,对OsLRR6的诱导表达特征进行分析(图2)。
3、OsLRR6基因突变水稻品系的获得及其对褐飞虱种群适合度的影响
将构建好的CRISPR/Cas9载体质粒(图3)电击转入农杆菌,鉴定无误后保存为工程农杆菌。以农杆菌转化法获得OsLRR6基因突变水稻植株,提取植物基因组DNA、测序、喷施苯达松以及qRT-PCR等方法筛选出K1和K3两个无T-DNA插入痕迹的纯合品系(图4)供后续试验使用。实验室环境下,种群适合度试验表明:与常规水稻植株相比,突变OsLRR6显著降低了褐飞虱产卵雌成虫的取食和产卵偏好(图5)、褐飞虱卵的孵化率(图6),提高了植株对褐飞虱的耐受性(图7)。田间试验表明:突变OsLRR6显著降低了田间褐飞虱和白背飞虱的种群数量(图8)。
图4中,a\b用于邓肯方差分析标记差异显著性;将每个时间点需要比较的三组数据(分别是WT、K1和K3)均值从大到小排列。例如0小时,WT品系对应的表达量最高,标记为a;K1品系对应的表达量第二高且与WT相比存在显著差异,因此标记为b;K3品系对应的表达量最低且与WT相比存在显著差异,因此标记为b,但是K3与K1相比不存在差异,所以K1和K3之间不需要用新的字母,最终K3标记为b。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明范围。
实施例1、OsLRR6基因的获得和序列分析
1)水稻叶鞘总RNA的提取、质量检测及cDNA第一链合成
2)以总cDNA第一链为模板,进行PCR反应,获得OsLRR6全长基因序列
OsLRR6-F1:5’-CATTGTGCCAACTGCCAAGG-3’;
OsLRR6-R1:5’-ACTCCCATCTCCCAAGGTGT-3’;
PCR扩增条件:95℃×4min→(98℃×15sec→60℃×40sec→68℃×50sec)×35循环→68℃×5min,得到特异PCR扩增产物见图1。
3)OsLRR6基因序列分析
将获得的PCR产物送南京金斯瑞公司进行测序,测序结果为序列表中的序列SEQID No.1。我们将此基因命名为OsLRR6。
实施例2、褐飞虱产卵雌成虫为害前后的OsLRR6表达特征分析
1)褐飞虱处理
在水稻的叶鞘基部固定一个圆筒形玻璃罩(直径4cm,高8cm,筒壁均匀分布24个直径为0.8mm的透气小孔),在玻璃罩内接入15头褐飞虱产卵雌成虫后,顶部以海绵密封。接入褐飞虱后在不同时间点剪取为害部位的外层叶鞘,立即浸入液氮,-80℃保存备用。以套入空玻璃罩的健康水稻叶鞘作为对照。
2)RNA的提取及表达特征分析
利用MiniBEST Plant RNA Extraction Kit(TaKaRa)试剂盒提取总RNA,并利用超微量蛋白核酸分光光度计(BioDrop)和甲醛凝胶变性电泳检测与评估所得RNA的浓度、纯度与质量。用PrimeScriptTMRT Master Mix(TaKaRa)反转录试剂盒将500ng总RNA反转录成cDNA,具体操作参照产品说明书。
荧光定量PCR(qRT-PCR)检测使用Premix Ex Taq[Probe qPCR](TaKaRa)酶预混液配制反应体系,使用CFX96TMReal-Time system(Bio-RAD)定量PCR仪检测荧光信号。以水稻OsACTIN基因(TIGR ID:LOC_Os03g50885)作为内参,对OsLRR6的诱导表达特征进行分析(图2)。具体反应体系及程序见产品说明书,定量PCR引物及探针如下:
OsACTIN-P:5’-CGTTTCCGCTGCCCTGAGGTCC-3’
OsACTIN-F:5’-GGACAGGTTATCACCATTGGT-3’
OsACTIN-R:5’-CCGCAGCTTCCATTCCTATG-3’
OsLRR6-P:5’-ACAACTCCGTCGTCCGCGTGG-3’
OsLRR6-F:5’-GTTCCATGTCACCTGCAACAAT-3’
OsLRR6-R:5’-CTGATAGACCTGCTAACCCCAAA-3’
实施例3、OsLRR6转基因品系的获得
1)人工合成权力要求3中所述的靶点序列的正义链和反义链(分别添加接头序列),退火合成靶序列双链,利用DNA亚克隆方法将其连入由浙江大学舒庆尧教授实验室改造的pHun4c12-Beli载体,获得pHun4c12-Beli-koLRR6突变表达质粒。并通过电击法,将pHun4c12-Beli-koLRR6质粒转入农杆菌LBA4404中,用于后续植物转化。人工合成的添加接头的靶点序列如下:
OsLRR6-F2:5’-ggcaCCAGGTGCAGGGATTTACA-3’
OsLRR6-R2:5’-aaacTGTAAATCCCTGCACCTGG-3’
2)用含有pHun4c12-Beli-koLRR6载体质粒的农杆菌侵染水稻愈伤组织。将共侵染后的愈伤放在含有潮霉素的NBDS培养基上筛选培养20天左右,待新的抗性愈伤长出后,将抗性愈伤从母体上剥离,转入新的筛选培养基NBDS2继代扩繁。将扩繁后的抗性愈伤转入分化培养基MS-RG培养2-3周。待其长出嫩芽后,切除多余愈伤组织并将嫩芽转入MS-RT培养基生根,分化出完整的T0代植株。
3)剪取T0代转基因植株少量叶片,提取基因组DNA并作为模板,用潮霉素鉴定引物Hyp-F/R筛选转基因阳性植株。再用引物koLRR6-seq-F/R扩增OsLRR6靶点前后产物并测序,用Chromas软件分析测序结果确定突变类型,筛选T0纯合突变植株。Hyp-F/R和koLRR6-seq-F/R引物分别如下:
Hyp-F:5’-GCCTGACCTATTGCATCTCCC-3’
Hyp-R:5’-GCTCCATACAAGCCAACCACG-3’
koLRR6-seq-F:5’-TGCCTTGGCTACACTTGTGA-3’
koLRR6-seq-R:5’-GTGACATGGCTTGTGAGCTG-3’
4)改造后的pHun4c12s-Beli载体的T-DNA部分插入了能够沉默水稻苯达松抗性基因的元件,即利用RNAi技术沉默苯达松抗性基因,因此组织培养后含有T-DNA的转基因植株对苯达松表现出易感。利用这一特性,将T0纯合品系繁种获得的T1水稻种子催芽长成高10cm左右秧苗时,对其叶片连续喷施1gL-1苯达松2-3天后,有T-DNA插入的突变品系因易感苯达松而枯死。为了确保存活的植株是无T-DNA插入的突变植株,再利用潮霉素鉴定引物Hyp-F/R判断是否仍含有潮霉素片段。电泳鉴定PCR产物未出现潮霉素条带的植株即确定为无T-DNA插入的T1代纯合突变品系,并用qRT-PCR的方法筛选转基因植株中OsLRR6转录水平显著降低的纯合品系,而后转入田间大量扩繁供后续试验使用。
实施例4、OsLRR6转基因突变品系的抗虫功能研究
1)雌成虫取食及产卵偏好性测定:在小塑料杯中分别固定1株常规水稻和1株突变体水稻(苗间距0.5cm),2株水稻叶鞘基部固定一个圆筒形玻璃罩,在玻璃罩内接入15头褐飞虱产卵雌成虫后,顶部以海绵密封。分别于接虫后1、2、4、8、12、24、48h观察并记录两株苗上的着虫数。48h后,去除所有褐飞虱,镜检每株苗上的产卵量并计算产卵分布比例。每个水稻品系处理设置10个生物学重复。
2)卵的孵化率测定:在单株水稻叶鞘基部固定一个圆筒形玻璃罩,向玻璃罩内接入15头BPH产卵雌成虫,然后用海绵将玻璃罩顶口密封。产卵12h后去除所有产卵雌成虫,每天观察并记录植株上孵化出的若虫数,直至无若虫孵出后,剪取水稻为害部位,镜检每棵植株上未孵化的卵量,计算并统计卵的孵化率。每个水稻品系处理设置10个生物学重复。
3)水稻耐害性测定:选取生长状况一致的水稻,每株接入15头褐飞虱产卵雌成虫,每个品系重复10次。每天观察水稻的生长情况并进行拍照。
上述实验均在温室(26±2℃;光照14h;湿度70%-75%)中进行。
4)田间试验:田间试验在浙江省湖州市长兴县泗安镇浙江大学长兴农业科技园(浙江大学转基因植物试验基地)中进行。试验田总面积约340m2(20m×17m),划分为9个小区,每个小区约27m2(6m×4.5m),且各小区间设有宽0.5m对照植株(WT)的保护行。试验田设计采用随机区组排列,将突变品系(K1和K3)和对照植株(WT)随机种植在9个小区中,每个品系设置3个重复。采用Z字形取样法,在每个小区内随机选择15个样点进行调查,记录每个样点(每丛水稻上)褐飞虱和白背飞虱的着虫数量。以15个样点的平均数作为该小区调查各项目的最终值,并统计分析。
结果表明:与常规水稻植株相比,突变OsLRR6显著降低了褐飞虱产卵雌成虫的取食和产卵偏好(图5)、褐飞虱卵的孵化率(图6),提高了植株对褐飞虱的耐受性(图7)。此外,田间应用OsLRR6突变植株也显著降低了褐飞虱和白背飞虱的种群数量(图8)。
实施例5、OsLRR6基因在水稻抗虫育种中的应用
1)以实施例3获得的OsLRR6基因突变的纯合品系水稻为材料,分析OsLRR6基因片段在水稻抗虫育种中的应用。
2)褐飞虱产卵雌成虫分别危害转基因苗与对照苗,如图7所示,13天后对照水稻基本枯死,而OsLRR6突变的转基因水稻仅外面的叶片枯黄。且如图8所示,田间应用OsLRR6突变的水稻植株显著降低了褐飞虱和白背飞虱的种群密度。可见OsLRR6基因片段可以很好的应用于抗稻飞虱的水稻抗虫育种。
序列表
<110> 浙江大学
<120> 水稻源抗虫相关基因OsLRR6及其编码产物与应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 848
<212> DNA
<213> 水稻(Oryza sativa)
<400> 1
cattgtgcca actgccaagg aactactcag gtttttcagt aatgggggct cattcttcag 60
cagcagcagc agctctgttc actggctttc ttgccttggc tacacttgtg agctgcaaca 120
ctgaaggtga cattctgtac gcgcaaaggc tggcatggaa ggaccccttc aatgtgctgc 180
agagttggga tccaaccctt gtaaatccct gcacctggtt ccatgtcacc tgcaacaata 240
acaactccgt cgtccgcgtg gatttggggt tagcaggtct atcaggtcct ctgattccac 300
agctgggagg actgagttac cttcagtacc ttgaactgta tgggaatgag ctgaatggat 360
caataccagc agcactgggc aacctgagca gtcttgtcag ccttgatctc cagggcaacc 420
tgctcactgg cgcgataccg gattcgctag gcgccattag caccctgcga aatctgaggt 480
tgtatgggaa caacctgact ggcaccatac cacaatcttt gggcagcctg acgagccttg 540
tcaaattgga gcttcagaag aattcattga gtggcaccat ccctgcttct ctcggcaaca 600
tcaagacatt ggagttgttg cgactgaaca aaaattcact taccggcaca gtgccaatgg 660
aagtcctctc ccttgtcctt gtcggcaact tgactgagct aaatgttgct ggaaacaatt 720
tggacggcac tgtcggatca actgggtgga gagtgactac catcattcag gacaatctga 780
agacctcagg ctgaggcaaa tatcaggagt caggatttgt tttaccacac accttgggag 840
atgggagt 848
<210> 2
<211> 250
<212> PRT
<213> 水稻(Oryza sativa)
<400> 2
Met Gly Ala His Ser Ser Ala Ala Ala Ala Ala Leu Phe Thr Gly Phe
1 5 10 15
Leu Ala Leu Ala Thr Leu Val Ser Cys Asn Thr Glu Gly Asp Ile Leu
20 25 30
Tyr Ala Gln Arg Leu Ala Trp Lys Asp Pro Phe Asn Val Leu Gln Ser
35 40 45
Trp Asp Pro Thr Leu Val Asn Pro Cys Thr Trp Phe His Val Thr Cys
50 55 60
Asn Asn Asn Asn Ser Val Val Arg Val Asp Leu Gly Leu Ala Gly Leu
65 70 75 80
Ser Gly Pro Leu Ile Pro Gln Leu Gly Gly Leu Ser Tyr Leu Gln Tyr
85 90 95
Leu Glu Leu Tyr Gly Asn Glu Leu Asn Gly Ser Ile Pro Ala Ala Leu
100 105 110
Gly Asn Leu Ser Ser Leu Val Ser Leu Asp Leu Gln Gly Asn Leu Leu
115 120 125
Thr Gly Ala Ile Pro Asp Ser Leu Gly Ala Ile Ser Thr Leu Arg Asn
130 135 140
Leu Arg Leu Tyr Gly Asn Asn Leu Thr Gly Thr Ile Pro Gln Ser Leu
145 150 155 160
Gly Ser Leu Thr Ser Leu Val Lys Leu Glu Leu Gln Lys Asn Ser Leu
165 170 175
Ser Gly Thr Ile Pro Ala Ser Leu Gly Asn Ile Lys Thr Leu Glu Leu
180 185 190
Leu Arg Leu Asn Lys Asn Ser Leu Thr Gly Thr Val Pro Met Glu Val
195 200 205
Leu Ser Leu Val Leu Val Gly Asn Leu Thr Glu Leu Asn Val Ala Gly
210 215 220
Asn Asn Leu Asp Gly Thr Val Gly Ser Thr Gly Trp Arg Val Thr Thr
225 230 235 240
Ile Ile Gln Asp Asn Leu Lys Thr Ser Gly
245 250
Claims (2)
1.一种水稻源OsLRR6基因在抗虫水稻育种中的应用;所述水稻源OsLRR6基因具有SEQID No.1的DNA序列;所述应用为降低水稻中水稻源OsLRR6基因的表达水平,增加水稻对褐飞虱和白背飞虱的抗性。
2.一种水稻源OsLRR6基因的DNA片段在抗虫水稻育种中的应用;所述DNA片段为基因OsLRR6全长DNA序列SEQ ID No.1中的第200到第219的碱基序列;所述应用为降低水稻中所述DNA片段的表达水平,增加水稻对褐飞虱和白背飞虱的抗性。
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