CN114438120B - 一种影响鳞翅目昆虫取食的植物内源基因及其蛋白 - Google Patents

一种影响鳞翅目昆虫取食的植物内源基因及其蛋白 Download PDF

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CN114438120B
CN114438120B CN202210169599.6A CN202210169599A CN114438120B CN 114438120 B CN114438120 B CN 114438120B CN 202210169599 A CN202210169599 A CN 202210169599A CN 114438120 B CN114438120 B CN 114438120B
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张悦
宋素胜
刘春琴
周璐璐
王庆雷
寇俊凤
吴娱
冯晓洁
刘福顺
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Cangzhou Academy Of Agriculture And Forestry Sciences
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Abstract

本发明提供了一种影响鳞翅目昆虫取食的植物内源基因及其蛋白。其中的植物内源其蛋白可以影响昆虫取食所述植物的量，所述蛋白的氨基酸序列如SEQ ID No.2所示。

Description

一种影响鳞翅目昆虫取食的植物内源基因及其蛋白
技术领域
本发明涉及一种影响鳞翅目昆虫取食的植物内源基因及其蛋白，特别是缺失或沉默该基因后的纯合子植物能够获得对鳞翅目昆虫的抗性。
背景技术
在农业生产中，害虫是影响作物产量和品质的重要原因之一，每年都会给农民造成巨大经济损失。鳞翅目包括蛾、蝶两类昆虫，属有翅亚纲、全变态类。该目为昆虫纲中仅次于鞘翅目的第2个大目，分布范围极广，以热带种类最为丰富。绝大多数种类的幼虫为害各类栽培植物，体形较大者常食尽叶片或钻蛀枝干。体形较小者往往卷叶、缀叶、结鞘、吐丝结网或钻入植物组织取食为害。成虫多以花蜜等作为补充营养，或口器退化不再取食，一般不造成直接危害。目前，针对作物害虫的防治主要依赖于化学农药，但化学农药具有刺激气味，对环境和人身健康都有严重威胁，所以从自然资源中挖掘天然杀虫材料越来越受到人们重视。在植物中寻找天然的抗虫基因或影响昆虫取食的基因，对于开发新型天然抗虫品系作物具有重要的理论和实践意义。
发明内容
本发明之一提供了一种植物内源蛋白在影响昆虫取食所述植物中的应用，所述蛋白的氨基酸序列如SEQ ID No.2所示。
在一个具体的实施方式中，通过抑制所述植物内源蛋白、失活所述植物内源蛋白、沉默所述植物内源蛋白，抑制所述植物内源蛋白的表达、沉默所述植物内源蛋白的表达以及降低所述植物内源蛋白的表达量中的一种使所述植物获得对昆虫的抗性。
在一个具体的实施方式中，所述昆虫为鳞翅目昆虫中的至少一种。
在一个具体的实施方式中，所述昆虫为灰翅夜蛾属昆虫中的至少一种。
在一个具体的实施方式中，所述昆虫为草地贪夜蛾和/或甜菜夜蛾。
在一个具体的实施方式中，所述植物为拟南芥。
本发明之二提供了编码如本发明之一中任意一项所述的应用中的植物内源蛋白的核酸在影响昆虫取食所述植物中的应用。
在一个具体的实施方式中，通过对所述核酸进行突变得到突变型植物获得对昆虫的抗性。
在一个具体的实施方式中，所述突变为插入突变、缺失突变、移码突变中的一种。
在一个具体的实施方式中，所述突变为插入突变或缺失突变。
在一个具体的实施方式中，所述突变为T-DNA插入突变。
在一个具体的实施方式中，所述突变型植物为突变型纯合子。
在一个具体的实施方式中，所述植物为拟南芥。
在一个具体的实施方式中，所述核酸的序列如SEQ ID No.1所示。
在一个具体的实施方式中，所述昆虫为鳞翅目昆虫中的至少一种。
在一个具体的实施方式中，所述昆虫为灰翅夜蛾属昆虫中的至少一种。
在一个具体的实施方式中，所述昆虫为草地贪夜蛾。
本发明的有益效果：
本发明首次发现在植物中存在的cznky2020002基因可以吸引鳞翅目昆虫幼虫，特别是草地贪夜蛾或甜菜夜蛾幼虫的取食，而将cznky2020002基因从植物中敲除或基因的部分序列缺失的纯合子则可以抑制昆虫幼虫的取食，进而提高植物对鳞翅目昆虫幼虫，特别是对草地贪夜蛾或甜菜夜蛾幼虫的抗虫性。因此，可以据此通过将植物中的cznky2020002基因敲除、沉默或缺失获得纯合子植株以达到抑制昆虫幼虫的取食，进而提高植物抗虫性的目的。
附图说明
图1为cznky2020002的基因结构。
图2显示了14天后取食cznky2020002和Col-0植株的草地贪夜蛾幼虫的平均单只虫重。
图3显示了14天后取食cznky2020002和Col-0植株的甜菜夜蛾幼虫的平均单只虫重。
具体实施方式
以下通过优选的实施案例的形式对本发明的上述内容作进一步的详细说明，但其不构成对本发明的限制。
如无特别说明，本发明的实施例中的试剂均可通过商业途径购买。
在ABRC(https://abrc.osu.edu/)官网订购拟南芥Col-0(野生型)、cznky2020002T-DNA插入型突变体cznky2020002(种子号：SALK_023552)。图1显示了cznky2020002的基因结构。如图1所示，在cznky2020002中，T-DNA插入在第6个外显子处。其中，cznky2020002的核酸序列如SEQ ID No.1所示，其蛋白的氨基酸序列SEQ ID No.2所示。
将拟南芥突变体cznky2020002种子点铺在MS培养基上，待拟南芥植株长至4-6片叶子时将其移栽到蛭石上生长(24℃；光照:黑暗＝16h:8h)，45天后各取24株分别提取突变体植株叶片的DNA。
1.cznky2020002突变型纯合子的筛选
以引物F1(SEQ ID No.3)、引物R1(SEQ ID No.4)，以及以突变体cznky2020002各植株的DNA为模板进行PCR扩增，用来鉴定突变体cznky2020002的各植株是否在cznky2020002基因上存在T-DNA插入；以引物F2(SEQ ID No.5)、引物R1(SEQ ID No.4)，以及以突变体cznky2020002各植株的DNA为模板进行PCR扩增，用来鉴定突变体cznky2020002的各植株是否存在cznky2020002基因。将F1/R1引物对扩增后的PCR扩增产物显示约600bp，且F2/R1引物对扩增后无PCR扩增产物的植株确定为cznky2020002纯合突变体植株，即突变型纯合子。
2.收获突变型纯合子的种子
待所筛选出的cznky2020002纯合突变体植株的种子成熟后，收取该cznky2020002纯合突变体的种子。
3.对草地贪夜蛾的抗虫试验
将纯合突变体cznky2020002的种子和野生型Col-0的种子分别点铺在MS培养基上，待植株长至4-6片叶子时，分别移栽至装满蛭石的花盆中(花盆尺寸为上直径7cm，下直径5cm，高8cm)，每盆移栽5株，每个基因型移栽10盆，将植株放置在24℃，光照:黑暗＝16h:8h条件下生长21天。将草地贪夜蛾初孵幼虫接到拟南芥植株叶片上，每盆接种20只，为1个重复，每个基因型3个重复。在花盆上覆盖透气性的PVC盖子，防治虫体逃逸。分别在第7天、第10天、第12天更换新的相应基因型的拟南芥植株，保证幼虫有充足的叶片可取食。整个试验持续14天。
在整个试验期间观察幼虫的取食情况，结果发现草地贪夜蛾幼虫取食cznky2020002植株的量少于野生型Col-0植株，这表明cznky2020002基因的存在能够吸引草地贪夜蛾幼虫取食该植物，而与野生型对照相比，cznky2020002基因缺失的纯合突变体更不易被草地贪夜蛾幼虫取食，从而表现出抗虫性。
在第14天，取出取食cznky2020002和Col-0植株的幼虫，分别单只称重，然后各处理取平均值。结果见图2。
从图2的结果可知，取食拟南芥突变型纯合子cznky2020002 14天后的草地贪夜蛾幼虫的虫重均显著低于取食拟南芥野生型Col-0的草地贪夜蛾幼虫的虫重(P<0.005)。这进一步表明cznky2020002基因的存在能够吸引草地贪夜蛾幼虫取食该植物，进而有利于草地贪夜蛾幼虫的生长发育；而与野生型对照相比，cznky2020002基因缺失的纯合突变体更不易被草地贪夜蛾幼虫取食，进而不利于草地贪夜蛾幼虫的生长发育，从而使有关植株表现出抗虫性。
4.对甜菜夜蛾的抗虫试验
操作同第3小节，不同之处仅在于将草地贪夜蛾替换为甜菜夜蛾。
甜菜夜蛾幼虫体重的结果见图3。
在整个试验期间观察幼虫的取食情况，结果发现甜菜夜蛾幼虫取食cznky2020002植株的量少于野生型Col-0植株，这表明cznky2020002基因的存在能够吸引甜菜夜蛾幼虫取食该植物，而与野生型对照相比，cznky2020002基因缺失的纯合突变体更不易被甜菜夜蛾幼虫取食，从而表现出抗虫性。
从图3的结果可知，取食拟南芥突变型纯合子cznky2020002 14天后的甜菜夜蛾幼虫的虫重均显著低于取食拟南芥野生型Col-0的甜菜夜蛾幼虫的虫重(P<0.005)。这进一步表明cznky2020002基因的存在能够吸引甜菜夜蛾幼虫取食植物，进而有利于甜菜夜蛾幼虫的生长发育；而与野生型对照相比，cznky2020002基因缺失的纯合突变体更不易被甜菜夜蛾幼虫取食，进而不利于甜菜夜蛾幼虫的生长发育，从而表现出抗虫性。
序列表
<110> 沧州市农林科学院
<120> 一种影响鳞翅目昆虫取食的植物内源基因及其蛋白
<130> LHA2060604
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3027
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 1
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ccttgtggcc tcatcaacgt tggcaatagc tgctttgcta atgttgtctt tcaatgcctt 1680
atgttcactc ctcctctgac gacatacttc ctccagcaat ttcattccag agcatgtaca 1740
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aagtctccac tgtcccctaa tggcttgcta tctcagctgc agaatattgg aatctttctt 1860
gggaatggca aggaagaaga tgcacatgaa ttccttaggt ttgttgtcga tacaatgcag 1920
tctgtgtgca ttaaggcatc tgaatatgat atgaccaaga gtagcaaatt agaagataca 1980
actcttattg gtttgacatt tgggggatac ctccggtcaa agattaaatg catgaaatgt 2040
caagtaaaat ctgagctgcg tgagaaaatg atggatctaa cggttgagat tgatggagat 2100
attagcaccc tggatgatgc tttgcgtcga tttacaagga ctgagatatt ggatggagag 2160
aacaaataca gatgtggcag ctgtaaatcc tatgagagag ccaaaaagaa gctgaaaata 2220
acagagcctc cgaatgtcct taccattgca ctaaaacgat tccaggcagg gaaattcggg 2280
aagctcaata agttgattag gtttccagaa actctagatt tggctccata tgtcagtggg 2340
ggaagtgaaa aatcacatga ctacaaactc tatggagtta ttgttcactt ggatgtaatg 2400
aatgcagcat tttctggcca ttatgtctgc tatattagaa accaaaataa gtggtacaag 2460
gctgatgata gcacggtagt aacttcggat gtggaaagga tcttgacaaa gggagcgtat 2520
atgttgttct acgcaaggtg cactccaacg cctccaagat tagcagtatg taccaaaact 2580
gaagcatcca ataaaaaaag cagagtgcca ctacctaagg ctaatgagaa aagtaccata 2640
tcccggtctg tgtctacttc aagtcctgag ttgtcttcca acacgcctgg tggtggtcga 2700
tccggcaata tacagtcatt ttattctagt ttccagaggc tgcagaagat tttagaagaa 2760
gactcggcga gtgacagttc atctctcttt gacagcaact cagatgaatg ctcttgtagc 2820
acagacagta caagcatgga cgactttgct gatttcattt ttggagatca tcagggacgg 2880
gctcacggac agtctgagac tccctcacca acatcatcgt cttcctcatc ttctcccccc 2940
ttcacaaggc gttcgccgct cagtcgttct tctccagaaa cttacggaac ctcaagacat 3000
cagttgccct tgggaggaga aaggtaa 3027
<210> 2
<211> 1008
<212> PRT
<213> 拟南芥(Arabidopsis thaliana)
<400> 2
Met Leu Leu Val Leu Asp Leu Gly Ile Ser Ser Leu Val Leu Val Val
1 5 10 15
Ser Leu Val Leu Pro Leu Ile Gly Leu Phe Val Arg His Lys Trp Arg
20 25 30
Val Ala Ala Gln Arg Arg Glu Glu Ile Arg Arg Leu Leu Ile His Ala
35 40 45
Ser Glu Glu Ala Ala Arg Ala Glu Leu Glu Ala Ser Val Glu Phe Ser
50 55 60
Ser Val Ala Val Ser Asn Val Phe His Cys Pro Val Cys Tyr Cys Leu
65 70 75 80
Ala Thr Thr Arg Cys Ser Arg Cys Lys Ala Val Arg Tyr Cys Ser Gly
85 90 95
Lys Cys Gln Ile Ile His Trp Arg Gln Gly His Lys Asp Glu Cys His
100 105 110
Pro Ala Ser Ile Val Tyr Asp Ser Glu Asp Glu Ser Asp Ser Asp Leu
115 120 125
Arg Leu Gly Glu Glu Asn Gly Gln Asn Thr Pro Glu Glu Thr Leu Leu
130 135 140
Val Gly Pro Glu Pro Val Thr Ile Pro Ile Gly Glu Ser Leu Leu Ser
145 150 155 160
Asn Arg Ala Arg Ser Pro Glu Asp Gly Asn Gly Asp Ile Ala Asp Asn
165 170 175
Lys Asp Asp Leu Ile Asp Lys Glu Glu Ala Val Ser Val Ala Glu Thr
180 185 190
Ser Gly Ser Ser Phe Ser Gly Phe Ser Ser Ser Pro Arg Asn Asp Ser
195 200 205
Gly Asp Glu Ile Ser Arg Cys Glu Ser Phe Ser Ser Ser Glu Ser Glu
210 215 220
Arg Ser Glu Ser Leu Leu Asp Ala His Val Ser Val Glu Pro Glu Asp
225 230 235 240
Thr Cys Phe Ser Thr Ile Glu Asp Ala Pro Ser Lys Leu Leu Ser Pro
245 250 255
Lys Phe Val His Leu Val Glu Ser Val Asp Asn Leu Ala Asn Leu Pro
260 265 270
Lys Leu Ser Val His Lys Pro Glu Asp Asp Ala Gly Gln Asn Gln Ser
275 280 285
Gln Ser Arg Ser Leu His Ser Leu Val Thr Asp Arg His Pro Val Ser
290 295 300
Ala Asp Pro Ser Leu Lys Ser Ser Asp Phe Trp Gly Thr Ala Leu Gly
305 310 315 320
Ser Ala Glu Arg Val Ser Asp Ser Cys Val Lys Ser Lys Ser Gly Arg
325 330 335
Pro Gly Asn Ser Ser Leu His Phe Ser Phe Gly Ser Gly Ser Ser Arg
340 345 350
Asp Thr Ser Ala Ala Lys Val Ser Glu Gln Arg Ser Ser Ile Leu Lys
355 360 365
Glu Ala Pro Arg Gly Thr Gly Tyr Ile Ser Asp Gly Val Asn Leu Arg
370 375 380
Glu Arg Asn Ala Lys Arg Phe Asp Glu Ala Glu Ile Ala Leu Pro Ile
385 390 395 400
Ser Ser Ser Thr Asp Ala Leu Ser Pro Leu Asp Ser Ser Asn Leu Ser
405 410 415
His Val Thr Leu Pro Lys Ser Lys Ser Ala Ser Ser Glu Asn Gly Ser
420 425 430
Met Leu Ala Pro Leu Lys Val Gly Glu Val Gln Leu Leu Ala Ser Lys
435 440 445
Ala Ser Asn Thr Lys Lys Cys Ala Asp Leu Met Lys His Ser Pro Leu
450 455 460
Gly Ala Lys Ser Val Arg Val Leu Asp His Gln Lys Gln Asn Gly Ala
465 470 475 480
Val Val Gln His Ile Asn Ser Leu His Gly Arg Ser Gly Leu Lys Ala
485 490 495
Ser Val Leu Lys Val Val Asp Gln Trp Thr Arg Pro Lys Ser Glu Asn
500 505 510
Glu Met Ala Gly Arg His Gly His Lys Gly Leu Phe Pro Tyr Glu Val
515 520 525
Phe Ala Lys Leu Tyr Thr Tyr Lys Ile Glu Phe Gln Pro Cys Gly Leu
530 535 540
Ile Asn Val Gly Asn Ser Cys Phe Ala Asn Val Val Phe Gln Cys Leu
545 550 555 560
Met Phe Thr Pro Pro Leu Thr Thr Tyr Phe Leu Gln Gln Phe His Ser
565 570 575
Arg Ala Cys Thr Lys Lys Glu Gln Cys Phe Thr Cys Gly Phe Glu Lys
580 585 590
Leu Val Val Lys Ala Lys Glu Glu Lys Ser Pro Leu Ser Pro Asn Gly
595 600 605
Leu Leu Ser Gln Leu Gln Asn Ile Gly Ile Phe Leu Gly Asn Gly Lys
610 615 620
Glu Glu Asp Ala His Glu Phe Leu Arg Phe Val Val Asp Thr Met Gln
625 630 635 640
Ser Val Cys Ile Lys Ala Ser Glu Tyr Asp Met Thr Lys Ser Ser Lys
645 650 655
Leu Glu Asp Thr Thr Leu Ile Gly Leu Thr Phe Gly Gly Tyr Leu Arg
660 665 670
Ser Lys Ile Lys Cys Met Lys Cys Gln Val Lys Ser Glu Leu Arg Glu
675 680 685
Lys Met Met Asp Leu Thr Val Glu Ile Asp Gly Asp Ile Ser Thr Leu
690 695 700
Asp Asp Ala Leu Arg Arg Phe Thr Arg Thr Glu Ile Leu Asp Gly Glu
705 710 715 720
Asn Lys Tyr Arg Cys Gly Ser Cys Lys Ser Tyr Glu Arg Ala Lys Lys
725 730 735
Lys Leu Lys Ile Thr Glu Pro Pro Asn Val Leu Thr Ile Ala Leu Lys
740 745 750
Arg Phe Gln Ala Gly Lys Phe Gly Lys Leu Asn Lys Leu Ile Arg Phe
755 760 765
Pro Glu Thr Leu Asp Leu Ala Pro Tyr Val Ser Gly Gly Ser Glu Lys
770 775 780
Ser His Asp Tyr Lys Leu Tyr Gly Val Ile Val His Leu Asp Val Met
785 790 795 800
Asn Ala Ala Phe Ser Gly His Tyr Val Cys Tyr Ile Arg Asn Gln Asn
805 810 815
Lys Trp Tyr Lys Ala Asp Asp Ser Thr Val Val Thr Ser Asp Val Glu
820 825 830
Arg Ile Leu Thr Lys Gly Ala Tyr Met Leu Phe Tyr Ala Arg Cys Thr
835 840 845
Pro Thr Pro Pro Arg Leu Ala Val Cys Thr Lys Thr Glu Ala Ser Asn
850 855 860
Lys Lys Ser Arg Val Pro Leu Pro Lys Ala Asn Glu Lys Ser Thr Ile
865 870 875 880
Ser Arg Ser Val Ser Thr Ser Ser Pro Glu Leu Ser Ser Asn Thr Pro
885 890 895
Gly Gly Gly Arg Ser Gly Asn Ile Gln Ser Phe Tyr Ser Ser Phe Gln
900 905 910
Arg Leu Gln Lys Ile Leu Glu Glu Asp Ser Ala Ser Asp Ser Ser Ser
915 920 925
Leu Phe Asp Ser Asn Ser Asp Glu Cys Ser Cys Ser Thr Asp Ser Thr
930 935 940
Ser Met Asp Asp Phe Ala Asp Phe Ile Phe Gly Asp His Gln Gly Arg
945 950 955 960
Ala His Gly Gln Ser Glu Thr Pro Ser Pro Thr Ser Ser Ser Ser Ser
965 970 975
Ser Ser Pro Pro Phe Thr Arg Arg Ser Pro Leu Ser Arg Ser Ser Pro
980 985 990
Glu Thr Tyr Gly Thr Ser Arg His Gln Leu Pro Leu Gly Gly Glu Arg
995 1000 1005
<210> 3
<211> 19
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 3
attttgccga tttcggaac 19
<210> 4
<211> 25
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 4
atataaagct catgtaaaac tctat 25
<210> 5
<211> 25
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 5
ttcattccag agcatgtgcg tcact 25

Claims (8)

1. 一种植物内源蛋白在影响昆虫取食所述植物中的应用，所述蛋白的氨基酸序列如SEQ ID No. 2所示；
通过抑制所述植物内源蛋白、失活所述植物内源蛋白、沉默所述植物内源蛋白，抑制所述植物内源蛋白的表达、沉默所述植物内源蛋白的表达以及降低所述植物内源蛋白的表达量中的一种使所述植物获得对昆虫的抗性；
所述昆虫为草地贪夜蛾或甜菜夜蛾。
2.根据权利要求1所述的应用，其特征在于，所述植物为拟南芥。
3.编码如权利要求1或2所述的应用中的植物内源蛋白的核酸在影响昆虫取食所述植物中的应用；
通过对所述核酸进行突变得到突变型植物获得对昆虫的抗性；
所述突变型植物为突变型纯合子；
所述昆虫为草地贪夜蛾或甜菜夜蛾。
4.根据权利要求3所述的应用，其特征在于，所述突变为插入突变、缺失突变、移码突变中的一种。
5.根据权利要求3所述的应用，其特征在于，所述突变为插入突变或缺失突变。
6.根据权利要求3所述的应用，其特征在于，所述突变为T-DNA插入突变。
7.根据权利要求3所述的应用，其特征在于，所述植物为拟南芥。
8. 根据权利要求3至7中任意一项所述的应用，其特征在于，所述核酸的序列如SEQ IDNo. 1所示。
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EP1059354A2 (en) * 1999-06-10 2000-12-13 Ceres Incorporated Sequence-determined DNA fragments and corresponding polypeptides encoded thereby
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EP1059354A2 (en) * 1999-06-10 2000-12-13 Ceres Incorporated Sequence-determined DNA fragments and corresponding polypeptides encoded thereby
CA2483494A1 (en) * 2002-04-25 2003-11-06 Emory University Semisynthetic protein-based site-directed probes for identification and inhibition of active sites, and methods therefor
AU2009208640A1 (en) * 2008-01-31 2009-08-06 National Institute For Biological Sciences Plants having altered growth and/or development and a method for making the same
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