CN107217061A - 水稻基因cyp71a1和5‑羟色胺在调控水稻植株抗虫性中的应用 - Google Patents
水稻基因cyp71a1和5‑羟色胺在调控水稻植株抗虫性中的应用 Download PDFInfo
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Abstract
本发明公开了水稻基因CYP71A1和5‑羟色胺在调控水稻植株抗虫性中的应用。本发明通过对γ射线诱变获得的突变体与野生型进行害虫抗性实验,发现基因CYP71A1突变的水稻植株相比野生型具有更高地抗虫性,验证了抑制基因CYP71A1表达在提高水稻植株抗虫性中的应用;与此同时,通过调节植株体内5‑羟色胺含量的变化,发现5‑羟色胺含量的降低可提高植株抗虫性,从而验证了抑制5‑羟色胺在提高水稻植株抗虫性中的应用。
Description
技术领域
本发明涉及植物基因工程和水稻分子育种领域,尤其涉及水稻基因CYP71A1和5-羟色胺在调控水稻植株抗虫性中的应用。
背景技术
水稻是一种重要的粮食作物,尤其是对亚洲国家而言,保证其稳定生产是十分重要的。褐飞虱是一种刺吸类昆虫,通过刺吸式口器刺入叶鞘吸收水稻的营养物质,其侵害可以引起植株生长缓慢,导致产量下降,甚至表现出“烧苗”(Hopperburn)症状,植株大面积死亡。此外,稻飞虱还会传播多种水稻病毒病。
目前,本领域对于稻飞虱的防治以化学防治为主,这导致用药成本增加、害虫抗药性上升、有害物质残留、环境污染等问题。
在褐飞虱抗性研究上,研究者已经挖掘了20多个抗性位点,但是克隆的基因并不多,目前只有Bph14,Bph26,Bph3,Bph18,Bph29被克隆。不同抗性基因的抗性谱和抗虫机制存在差异。如携带Bph14的水稻品种表现出抗生性,缩短褐飞虱取食时间、抑制其生长发育等;携带来源于南亚的一个品种RHT的抗性基因Bph3,由3个凝集素受体激酶组成。
尽管较多的QTL位置被发掘,但从克隆的基因数量来看,还是较少的,其背后的抗性机制也并不清楚。因此,抗褐飞虱水稻种质资源,尤其是广谱性抗性材料的发掘是十分紧迫的。
水稻基因CYP71A1属于单加氧酶P450家族基因,能够以色胺为底物,催化其芳环5'位置进行羟化反应,形成5羟色胺。因此,CYP71A1又名T5H(Tryptamine 5Hydroxylation)。
发明内容
本发明提供了一种水稻基因CYP71A1和5-羟色胺在调控水稻植株抗虫性中的应用,该基因的缺失能够提高水稻植株的抗虫性。
水稻基因CYP71A1在调控水稻植株抗虫性中的应用,其中,所述水稻基因CYP71A1(LOC_Os12g16720)的核苷酸序列如SEQ ID NO.1所示。
具体地,为了提高水稻植株的抗虫性,可以采用以下方法:
(1)通过化学物质抑制基因CYP71A1表达或者通过RNAi抑制基因CYP71A1的转录;(2)对基因CYP71A1进行基因突变,例如:γ射线或者EMS处理诱发变异,或者CRISPR基因编辑技术等。
本发明提供了一种提高水稻抗虫性的方法,包括:采用CRISPR技术将水稻基因组中基因CYP71A1进行定点突变,获得基因CYP71A1沉默的水稻植株。
针对基因CYP71A1对虫害的适用范围,本发明提供了褐飞虱、白背飞虱和螟虫的验证实验;发现与基因CYP71A1突变后的水稻植株相比,褐飞虱、白背飞虱和螟虫更偏向于取食野生型水稻植株。
此外,本发明还通过实验验证了5-羟色胺在调控水稻植株抗虫性中的应用,发现5-羟色胺的降低有利于水稻植株抗虫性的提高;所以采用抑制植株体内5-羟色胺含量的化学物质同样也可以降低水稻植株的抗虫性。
与现有技术相比,本发明具有以下有益效果:
本发明通过对田间获得的突变体与野生型进行害虫抗性实验,发现基因CYP71A1突变的水稻植株相比野生型具有更高地抗虫性,验证了抑制基因CYP71A1的表达在提高水稻植株抗虫性中的应用;与此同时,通过调节植株体内5-羟色胺含量的变化,发现5-羟色胺含量的降低可提高植株抗虫性,从而验证了抑制5-羟色胺在提高水稻植株抗虫性中的应用。
附图说明
图1为实施例1中野生型水稻植株嘉浙B和突变体植株嘉浙LM对褐飞虱抗性的比较;
其中,a为将野生型水稻和突变体共同培养并供褐飞虱自由取食后的结果图;b为褐飞虱取食选择性实验,将15头成虫放进密闭玻璃杯罩内,不同的时间点观察野生型和突变体植株上的数目;c为b实验中的褐飞虱在48小时之后,便会在水稻茎秆产卵,野生型和突变体植株上的产卵数统计结果。
图2为实施例1中褐飞虱二龄幼虫接于野生型水稻植株嘉浙B和突变体植株嘉浙LM上15天后幼虫存活率。
图3为实施例1中野生型水稻植株嘉浙B和突变体植株嘉浙LM对白背飞虱抗性的比较;
其中,a为白背飞虱的选择性取食实验;b为a中白背飞虱取食48小时之后在野生型和突变体上的产卵数统计结果。
图4为实施例1中野生型水稻植株嘉浙B和突变体植株嘉浙LM对螟虫抗性的比较;
其中,a为一直以野生型或者突变体水稻植株取食的二化螟在21天和28天后的发育大小;b为同一时间点上,各个龄期的螟虫数目统计分析结果;c为取食野生型植株的螟虫在不同天数下幼虫化成蛹的数量变化图,其中,大约历时36.94天,幼虫化成蛹的数量达到峰值;d为取食突变体植株的螟虫在不同天数下幼虫化成蛹的数量变化图,其中,大约历时47.46天,幼虫化成蛹的数量达到峰。
图5为实施例1中大田种植环境下野生型水稻植株嘉浙B和突变体植株嘉浙LM对二化螟幼虫抗性的比较;
其中,a为田间生测实验,每株水稻接种一头二化螟幼虫;b为每隔十天统计死亡的水稻植株,包括枯心和白穗。
图6为实施例2中不同水稻植株体内5-羟色胺的本底水平以及褐飞虱侵害植株8小时后植株体内5-羟色胺的含量。
图7为实施例2中不同水稻植株种植于不同浓度梯度的5-羟色胺营养液中,褐飞虱选择性取食实验;
其中,a为突变体植株内添加5-羟色胺回补后,取食的褐飞虱数量变化情况;b为往不同抗性材料中添加5-羟色胺后,取食的褐飞虱数量变化情况。
图8为实施例2中野生型水稻植株嘉浙B和突变体植株嘉浙LM各组织部位的5-羟色胺含量以及回补5-羟色胺后突变体植株体内5-羟色胺含量变化曲线;
其中,a为野生型和突变体植株根部组织中5-羟色胺的含量变化情况;b为野生型和突变体植株叶鞘组织中5-羟色胺的含量变化情况;c为野生型和突变体植株叶片组织中5-羟色胺的含量变化情况;d为通过往样品中添加5-羟色胺,确认5-羟色胺的特异峰;e为野生型材料中,各个组织部位5-羟色胺含量的比较;f为往突变体培养液中添加5-羟色胺后植株内5-羟色胺的含量。
图9为实施例2中褐飞虱侵害突变体植株嘉浙LM后,褐飞虱体内5-羟色胺含量的变化曲线。
图10为实施例3中基因沉默植株CYP71A1-KO与野生型植株锡稻一号对褐飞虱抗性的比较。
图11为实施例3中在不同种植区域种植下突变体与野生型植株的田间实验比较。
具体实施方式
在田间水稻种植时,发现一个突变体,该突变体分蘖期阶段的叶片上会形成类似病斑(褐色斑点)的表型,然而在室内水培种植时,该突变体的表型又与正常水稻植株无差异。虽然造成上述现象的原因还不清楚,但我们通过图位克隆发现,该突变体的基因CYP71A1中有一个G的缺失,并导致突变体丧失五羟色胺合成能力。通过实验发现,该基因的缺失会影响水稻的抗虫性,具体实验内容如下。
其中,下列实验提及的害虫有褐飞虱、白背飞虱和螟虫。褐飞虱是怀有卵的雌性飞虱,其具有非常强的攻击性,由浙江大学娄永根教授提供;白背飞虱由由浙江大学娄永根教授提供;螟虫是由浙江大学昆虫所的叶恭银教授提供。水稻品种“嘉浙B”是一个用于三系杂交水稻的保持系材料,来自嘉兴农科院;水稻品种“锡稻一号”由浙江大学舒庆尧教授提供。
实施例1选择性实验
本实施例以水稻突变体嘉浙LM(Jiazhe lesion mimic)和野生型嘉浙B为材料进行实验;其中,突变体嘉浙LM是通过采用350Gy 60Co辐照诱变野生型水稻嘉浙B获得;经检测,该突变体的基因组中基因CYP71A1有一个G的缺失。
1、褐飞虱抗性实验
a)取食选择性和产卵量:将野生型水稻和突变体水培于同一培养容器中,供褐飞虱自由取食(结果如图1a所示)。将野生型水稻和突变体水培于同一培养容器中,并在水稻的茎秆部位用透气玻璃罩(直径4厘米,高度8厘米)围住(两种植株在同一玻璃罩内),往透气玻璃罩内投入15头怀卵褐飞虱雌虫。在投入后的1,2,4,8,12,24和48小时,对上述两株植株上的褐飞虱数量进行计数,共进行十组重复(结果如图1b所示)。计数结束后,收集所有植株,在显微镜下对卵的数目进行计数,同时统计卵的孵化率(结果如图1c所示)。
b)采食量:将野生型与突变体在相同培养环境下进行分开水培,并均在水稻基部用封口膜紧密包裹,封口处各投放一头褐飞虱,24小时之后,对投放飞虱前后封口处水稻茎秆进行称重,获得质量差(即蜜露的量),共进行20组重复。
c)幼虫存活率:分别将15头褐飞虱的二龄幼虫接于野生型植株和突变体植株上,每天观察存活的数目,直至处理后15天。结果如图2所示。
由图1a可知,通过褐飞虱的自由取食选择15天之后,野生型材料嘉浙B几乎全部死亡,植株萎缩,然而其突变体嘉浙LM还保持绿色,能够继续生长。
为进一步进行褐飞虱选择性分析,将突变体和野生型放置在同一玻璃罩子内,每隔一定时间统计植株上的褐飞虱数量。实验发现,褐飞虱明显偏向于在野生型植株上取食,例如在侵害12小时之后,野生型植株上的褐飞虱数量是突变体植株上的1.55倍(图1b)。
选择性的差异,势必导致产卵数量的差异。48小时之后,我们对上述实验的每株水稻上的卵数进行统计,结果显示,野生型嘉浙B上卵的数量明显多于突变体嘉浙LM(图1c)。
此外,对褐飞虱在水稻上的取食量,也就是蜜露的含量进行了调查。蜜露就是昆虫的排泄物,其排泄的量越大在一定程度上也就意味着取食的量也越大。
与野生型相比,突变体上的蜜露含量也有30%的减少。以上数据表明,褐飞虱并不喜欢在突变体上取食。并且从图2可见,突变体严重影响了若虫的存活率。
2、白背飞虱抗性实验
同样从取食选择性和产卵量方面进行实验,实验方法与褐飞虱相同,结果如图3所示。
由图3可见,白背飞虱更倾向于在野生型上取食、产卵(如图3)。
3、螟虫抗性实验
A)将野生型与突变体的茎秆饲喂10头螟虫若虫,每隔7天换一次新鲜茎秆,直至螟虫发育成熟蛹化。考察螟虫的整个生长周期,从幼虫,蛹化,羽化到产卵量,统计最终蛹的数目和化蛹时间,结果如图4所示。
取食于突变体上的二化螟出现了发育延缓的症状,体态较小(图4a)。当野生型上的幼虫已经进入三龄的时候,突变体上的二化螟大多数还处于二龄期(图4b)。纵观整个发育阶段,最终大多数野生型植株上的成虫都会化成蛹,大约需要36.94天,而突突变体上的二化螟需要47.46天,成蛹数也较少(图4c,d)。
B)对田间种植的每株野生型和突变体接一头二化螟幼虫,每十天观察一次枯心和白穗的数目,结果如图5所示。
由图5可见,接二化螟幼虫的第二十天突变体群体中的白穗和枯心数明显少于野生型。
实施例2 5-羟色胺对水稻抗虫性的影响
为了探究突变体对褐飞虱的抗性是否与5-羟色胺的缺失有关,进一步测定已知携带有抗性基因的水稻材料以及褐飞虱敏感材料的5-羟色胺含量。
本实施例中植株体内5-羟色胺含量的测定方法,具体如下:
(1)样品预处理:五克叶片,茎秆或者根部组织在液氮中充分研磨至粉末,加入9mL甲醇,10℃下,180rpm摇床混匀,充分浸提30min,13,500×g,10℃离心15min,可以抽出8mL的上清液,按照1:4的比例,加入2mL蒸馏水,混匀。C18固相萃取小柱用三倍柱床体积的甲醇活化,之后用蒸馏水冲洗。将已加水的样品过小柱,流出液回收;再用10mL 80%的甲醇冲洗,冲洗出来的溶液也回收,与之前的流出液合并;最后,旋转蒸发,加入500μL 50%的甲醇溶解待测。
(2)高效液相色谱(HPLC)测定:分离柱为C18小柱,流动相为10%甲醇、0.3%三氟乙酸,流速控制在0.8mL/min,流动相分离30分钟之后,用100%甲醇冲洗5min,再用流动相跑平基线10min,以保证柱子中的其余杂质冲洗干净。检测器波长设置为280nm。
本实施例采用以下四种褐飞虱抗性材料:Mudgo(携带抗性基因BPH1)从浙江大学昆虫所娄永根教授实验室获得;RHT和IR56(两者均携带抗性基因BPH3)从中国水稻所傅强教授实验室获得;B5(携带抗性基因BPH14,BPH15)从华中农业大学牟同敏实验室获得。褐飞虱敏感材料采用以下两种:TN1(一个褐飞虱敏感材料,取自浙大农学院昆虫所娄永根课题组)和嘉浙B。
测定上述六种水稻植株体内5-羟色胺的本底水平,并用褐飞虱侵害植株,8小时后测定植株体内5-羟色胺的含量。
由图6可知,两种褐飞虱敏感材料(TN1和嘉浙B)有着较高的本底表达量,同时能被褐飞虱侵害诱导;而其余四个抗性材料(Mudgo,IR56,RHT,B5分别携带抗性基因Bph1,Bph3,Bph3,Bph14/15)的本底水平都很低,几乎不被褐飞虱侵害诱导(图6)。可见,抗性材料都反映出更趋向于低水平的5-羟色胺。
用5-羟色胺回补实验进一步验证上述结果。将植株种植于不同浓度梯度(0,50,100,200,500μM)的5-羟色胺营养液中,5-羟色胺通过根部吸收进入植株的叶鞘组织中。
由图7可知,当突变体植株营养液中5-羟色胺的浓度上升到300μM浓度时,褐飞虱的取食更加偏向于突变体,可见此浓度下能使原本5-羟色胺缺失的突变体得到恢复,从抗性变得敏感。同样,300μM浓度下也可以使得抗性水稻材料变得敏感。因此,低水平5-羟色胺更有助于提高褐飞虱的抗性,甚至是在基因组背景不同的水稻中,上述结论也适用。
此外,测定野生型和突变体植株各组织部位的5-羟色胺含量,并通过回补实验外源添加5-羟色胺。
从图8可知,突变体植株中根部的含量最高,叶鞘最低;外源添加5-羟色胺12小时后突变体中的5羟色胺含量能够恢复到野生型的本底水平。
用褐飞虱侵害植株,测定褐飞虱体内5-羟色胺的含量,结果如图9所示。褐飞虱取食12个小时后,取食于野生型植株上的虫体内5-羟色胺含量有28.54ng/g,而突变体上的褐飞虱体内5-羟色胺只有5.3ng/g。
实施例3
为进一步验证实施例1所得结论,我们进一步通过CRISPR技术,将水稻品种“锡稻一号”中的基因CYP71A1进行定点突变,获得基因CYP71A1沉默植株,即水稻植株CYP71A1-KO。
具体步骤如下:
根据基因序列,设计靶点序列为5'-TGGTCGCGTTGAGGAGGA GC-3',与CRISPR载体pHun4c12连接,转入农杆菌。经农杆菌介导转化之后,获得T0代植株,通过测序发现突变体。导致距离起始密码子83处插入一个“A”,从而形成移码,造成突变。经繁种,获得F1代,用于实验。
将野生型“锡稻一号”和基因沉默植株CYP71A1-KO进行采食选择性实验(步骤同实施例1)。由图10可知,基因沉默之后的植株也表现出抗性,褐飞虱并不喜欢在上面取食。因此,证实了基因CYP71A1的突变有助于获得对褐飞虱的抗性。
实施例4
田间实验证明突变体在农业生产中也能发挥较好的作用。将野生型和突变体种植于嘉兴和三亚两个地方,并统计植株上的飞虱量,同样发现突变体群体中的飞虱数量减少(图11)。
此外,我们进一步在田间接二化螟来检测突变体植株是否对螟虫有着较好的抗性。通过统计枯心和白穗的数目,同样证实能够提高对螟虫的抗性。(图5b)。
SEQUENCE LISTING
<110> 浙江大学
<120> 水稻基因CYP71A1和5-羟色胺在调控水稻植株抗虫性中的应用
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 2026
<212> DNA
<213> 水稻
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gttcatacca acacatctcc attgctgctt aagtttcttg gaccaaacgt gcaccccaag 60
tgttcgacga tatggagctc accatggcgt cgacgatgtc gctcgcgctg ctcgtgctct 120
ccgcggcgta cgtgttggtc gcgttgagga ggagccggtc gtcgtcgtca aagccacggc 180
ggctgccgcc gtcgccgccg gggtggccgg tgatcgggca cctccacctc atgtccggca 240
tgccgcacca cgcgctggcc gagctggcgc gcaccatgcg cgcgccgctg ttccggatgc 300
ggctggggag cgtgccggcg gtggtgatct ccaagccgga cctcgcccgc gccgcgctca 360
ccaccaacga cgccgcgctg gcgtcgcggc cgcacctgct ctccggccag ttcctgtcgt 420
tcggctgctc cgacgtgacg ttcgcgccgg cggggccgta ccaccggatg gcgcgccgcg 480
tggtggtgtc ggagctcctg tcggcgcgtc gcgtcgccac gtacggcgcc gtcagggtca 540
aggagctccg ccgcctgctc gcgcacctca ccaagaacac ctcgccggcg aagcccgtcg 600
acctcagcga gtgcttcctc aacctcgcca acgacgtgct ctgccgcgtc gcgttcggcc 660
gccggttccc gcacggcgag ggcgacaagc tcggcgcggt gctcgccgag gcgcaggacc 720
tcttcgccgg gttcaccatc ggcgacttct tccccgagct cgagcccgtc gccagcaccg 780
tcaccggact ccgccgccgc ctcaagaagt gcctcgccga cctccgcgag gcctgcgacg 840
tgatcgtgga cgaacacatc agcggcaacc gccagcgcat ccccggcgac cgcgacgagg 900
acttcgtcga cgtcctcctc cgcgtccaga aatcccccga cctcgaggtc cccctaaccg 960
acgacaatct caaggccctc gtcctggtac gccattaata cccaccatta aaactctcga 1020
attgactcgc cgccattgtt gctcctcctc tgatcttgac atgacgtgac aacaaccacc 1080
aggacatgtt cgtcgccggc acggacacca cgttcgcgac gctggagtgg gtgatgacgg 1140
agctagtccg ccacccacgg atcctcaaga aggcgcagga ggaggtccgg cgagtcgtcg 1200
gcgacagcgg ccgcgtcgag gagtcccacc tcggcgagct ccactacatg cgcgccatca 1260
tcaaggagac gttccggctg cacccggcgg tgccgttgct agtgccgcgc gagtccgtcg 1320
cgccgtgcac gctgggcggc tacgacatcc cggcgaggac gcgggtgttc atcaacacgt 1380
tcgccatggg gcgcgacccg gagatctggg acaacccgct ggagtactcg ccggagaggt 1440
tcgagagcgc cggcggcggc ggcgagatcg acctcaagga cccggactac aagctgctgc 1500
cgttcggcgg cgggcggcga gggtgccccg gctacacgtt cgcgctcgcc accgtgcagg 1560
tgtcgctcgc cagcttgctc taccacttcg agtgggcgct gcccgccggc gtgcgcgccg 1620
aggacgtcaa cctcgacgag acgttcggcc tcgccacgag gaagaaggag ccgctcttcg 1680
tcgccgtcag gaagagcgac gcgtacgagt ttaagggaga ggagcttagt gaggtttaag 1740
ttaaattaag taatgattag cgatagatag atttttatta tttttattat gttttgggat 1800
aataataagg gttaaagttt tgtgcttgct aagtaacctg gaggctggag ctggtaatat 1860
tgtgactaag cagagaagca agcctgtgtg atttcgatgt aaaagtacag gatatggtgc 1920
ctaatgaata aaagaagtca ataagtggcg tatttctctt gtaacttgta agctatatcg 1980
aattttaaac tttagagatt aatttatggt tctttttgtt atgatt 2026
<210> 2
<211> 20
<212> DNA
<213> 人工序列
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tggtcgcgtt gaggaggagc 20
Claims (4)
1.水稻基因CYP71A1在调控水稻植株抗虫性中的应用,其特征在于,所述水稻基因CYP71A1的核苷酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的应用,其特征在于,所述抗虫性为对褐飞虱、白背飞虱或螟虫的抗性。
3.5-羟色胺在调控水稻植株抗虫性中的应用。
4.一种提高水稻植株抗虫性的方法,其特征在于,包括:采用CRISPR技术将水稻基因组中基因CYP71A1进行定点突变,获得基因CYP71A1沉默的水稻植株。
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