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CN113416736B - 抗病基因Sw-5b人工改良突变体及其应用 - Google Patents

抗病基因Sw-5b人工改良突变体及其应用 Download PDF

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CN113416736B
CN113416736B CN202110544417.4A CN202110544417A CN113416736B CN 113416736 B CN113416736 B CN 113416736B CN 202110544417 A CN202110544417 A CN 202110544417A CN 113416736 B CN113416736 B CN 113416736B
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陶小荣
黄海宁
李佳
黄申
戴婧
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Abstract

抗病基因Sw‑5b人工改良突变体及其应用，该突变体为33位亮氨酸突变成脯氨酸，319位赖氨酸突变成谷氨酸，927位精氨酸突变成丙氨酸或谷氨酰胺，其余氨基酸位点保持不变，且三个氨基酸位点同时突变。本发明获得的Sw‑5bL33P/K319E/R927A与Sw‑5bL33P/K319E/R927Q两种突变体，该突变体对TSWV野生型及两种病毒田间变异株系TSWVC118Y、TSWVT120N均具有抗病性，为TSWV田间抗性突破变异株系的防控提供了有效的抗病材料。

Description

抗病基因Sw-5b人工改良突变体及其应用
技术领域
本发明属于植物基因工程领域，具体涉及抗病基因Sw-5b人工改良突变体及其应用。
背景技术
TSWV是布尼亚病毒目、番茄斑萎病毒科、正番茄斑萎病毒属的代表种。TSWV侵染范围广，能够侵染80个不同科的800多种植物。TSWV在番茄叶片上导致黑色斑点的出现，同时也会导致叶片尖端枯死，单侧生长及发育迟缓等症状。TSWV在侵染早期可能会导致番茄无法结实；在侵染晚期导致果实上出现同心圆的斑点。
目前，防治TSWV最为有效的手段是选育抗病品种。来源于秘鲁番茄的抗病基因Sw- 5b对TSWV具有良好的抗病作用。除TSWV外，Sw-5b对正番茄斑萎病毒属中的番茄褪绿病毒、凤仙花坏死斑萎病毒等美洲型病毒也具有良好的抗病毒效果。
随着携带Sw-5b抗病基因的抗病品种长期推广使用，田间逐渐产生能够克服Sw-5b介导的抗病性的TSWV变异株系（以下简称抗性突破型病毒株系）。2003年，抗性突破型病毒株系首次在西班牙田间发现，之后陆续在意大利、美国加利福尼亚州等地出现。其中，2016年，在美国加利福尼亚州的TSWV抗性突破型株系发病率约为50-80%，造成严重的经济损失。2011年Carmelo Lopez等研究发现TSWV抗性突破型株系主要分成两类，一类是TSWV移动蛋白NSm的118位半胱氨酸突变成络氨酸，即NSmC118Y；另一类是TSWV移动蛋白NSm的120位苏氨酸突变成天冬氨酸，即NSmT120N。目前，对于这两种抗性突破型病毒株系并没有有效的防治手段。由于抗番茄斑萎病毒属病毒的作物种质资源与品种匮乏，因此通过人工筛选手段对抗病基因Sw-5b进行改良设计获取新的抗病基因资源显得尤为重要。
结合当前抗病基因Sw-5b对TSWV NSm识别机制和抗病基因人工改良设计方法的前沿研究进展，通过对Sw-5b进行精确地人工分子设计和改良，赋予该抗病基因对番茄斑萎病毒田间变异株系新的抗病性。
发明内容
解决的技术问题：本发明针对上述技术问题，提供抗病基因Sw-5b的人工改良突变体及其应用。两种Sw-5b突变体（Sw-5bL33P/K319E/R927A与Sw-5bL33P/K319E/R927Q）对TSWV 野生型及两种病毒田间变异株系TSWVC118Y、TSWVT120N均具有抗病性。
技术方案：抗病基因Sw-5b人工改良突变体， 33位亮氨酸突变成脯氨酸，319位赖氨酸突变成谷氨酸，927位精氨酸突变成丙氨酸或谷氨酰胺，其余氨基酸位点保持不变，且三个氨基酸位点同时突变。
上述抗病基因Sw-5b人工改良突变体之一，氨基酸序列如SEQ ID NO.1所示。
上述抗病基因Sw-5b人工改良突变体之二，氨基酸序列如SEQ ID NO.2所示。
一种编码基因，其编码上述突变体。
含有上述编码基因的载体。
含有上述载体的重组菌或重组细胞。
上述抗病基因Sw-5b人工改良突变体在培育抗TSWV野生型、TSWVC118Y和TSWVT120N植物中的应用。
上述植物为番茄。
有益效果：本发明获得Sw-5bL33P/K319E/R927A与Sw-5bL33P/K319E/R927Q两种突变体，该突变体对TSWV 野生型及两种病毒田间变异株系TSWVC118Y、TSWVT120N均具有抗病性。本发明为TSWV田间抗性突破变异株系的防控提供了有效的抗病材料。
附图说明
图1为对Sw-5bR927A SD功能域进行随机突变流程图；
图2为Sw-5bL33P/K319E/R927A与Sw-5bL33P/K319E/R927Q与NSmWT、NSmC118Y、NSmT120N瞬时浸润本氏烟叶片HR症状图；
图3为Sw-5bL33P/K319E/R927A与Sw-5bL33P/K319E/R927Q转基因本氏烟对TSWV 野生型及两种病毒田间变异株系TSWVC118Y、TSWVT120N具有良好的抗病性；
图4为图3植物系统叶中TSWV积累量的检测结果。
图5为两个Sw-5b突变体（Sw-5bL33P/K319E/R927A与Sw-5bL33P/K319E/R927Q）氨基酸序列示意图。
具体实施方式
下面的实施例可使本专业技术人员更全面地理解本发明，但不以任何方式限制本发明。在不背离本发明精神和实质的情况下，对本发明策略、方法、步骤或条件所作的修改和替换，均属于本发明的范围。
实施例1：对Sw-5bR927A SD功能域进行随机突变
关于Sw-5bR927A突变体库的构建与Sw-5bR927A突变体筛选的策略（图1），简言之，通过Mn-dITP PCR的方法扩增大量发生碱基突变的Sw-5b SD PCR产物，具体操作如下：PCR扩增采用通用引物对 (正向引物 ： 5’- ATGGCTGAAAATGAAATTGAGGAAATGTTA -3’ ，反向引物：5’- GGGGAACTGAGATGGAGTAAATTTTAAGGA -3’)。在第一轮PCR扩增时，PCR 反应体系（50μL）：10×PCR缓冲液5 μL，MnCl2（1 mM）2 μL，MgCl2（25 mM）4 μL， dNTP（10 mM）1 μL，引物（10 μM）各1 μL，DNA模板（25 ng）4 μL，Taq DNA polymerse（5 U）1 μL，双蒸水 31 μL ；PCR扩增条件为：94℃预变性3 min，94℃变性1 min，51℃退火1 min，72℃延伸1min15s，循环20次，72℃后延伸10 min。在第二轮PCR扩增时，PCR 反应体系（50 μL）：10×PCR缓冲液5 μL，dITP（10 mM）2 μL，MgCl2（25 mM）4 μL， dNTP（10 mM）1 μL，引物（10 μM）各1 μL，DNA模板（第一轮PCR产物）2 μL，Taq DNA polymerse（5 U）1 μL，双蒸水 33 μL ；PCR扩增条件为：94℃预变性3 min，94℃变性1 min，51℃退火1 min，72℃延伸1min15s，循环30次，70℃后延伸10 min。同时扩增p2300-Sw-5bR927A△SD PCR产物，具体操作如下：PCR扩增采用通用引物对(正向引物：5’- TCCTTAAAATTTACTCCATCTCAGTTCCCC-3’ ，反向引物：5’-TAACATTTCCTCAATTT
CATTTTCAGCCAT -3’)。PCR 反应体系（50 μL）：2 × Phanta Max缓冲液25 μL，dNTP Mix（10 mM）1 μL，引物（10 μM）各2 μL，DNA模板2 μL，Phanta Max Super-FidelityDNA Polymerase 1 μL，双蒸水 17 μL ；PCR扩增条件为：95℃预变性3 min，95℃变性15s，58℃退火15s，72℃延伸6min30s，循环35次，72℃后延伸10 min。。将回收的Sw-5b SD PCR产物与回收的p2300-Sw-5bR927A△SD PCR产物进行同源重组，同源重组体系（20 μL）：5×CE IIBuffer 4 μL，线性化克隆载体（p2300-Sw-5bR927A△SD PCR）240 ng，插入片段扩增产物（Sw-5b SD）48 ng，Exnase II 2 μL, 双蒸水补齐至20 μL即可。将配置完成的同源重组体系置于37℃反应30 min。待反应完成后，立即将反应管置于冰水浴中冷却5 min。之后将同源重组产物进行电击转化，得到大量嵌合型的Sw-5b R927A突变体。
实施例2：筛选能够同时识别TSWV NSmWT、NSmC118Y、NSmT120N的Sw-5bR927A突变体
为了筛选能够与TSWV NSmWT、NSmC118Y或NSmT120N产生HR的Sw-5bR927A突变体，本发明对2036个Sw-5bR927A突变体进行验证，具体操作如下，挑取嵌合型Sw-5b R927A突变体单克隆于3-4 mL LB液体培养基（1% (w/v) tryptone, 0.5% (w/v) 酵母提取物, and 1% (w/v)NaCl），在28℃恒温培养箱220 rpm培养24 h。将菌液置于2 mL离心管中，6000 rpm离心10min；弃去上清，使用农杆菌处理液（(10 mM MES pH 5.6, 10 mM MgCl2, and 150 mM 乙酰丁香酮)悬起沉淀，OD600调至0.5即可。将Sw-5bR927A突变体与NSmWT、NSmC118Y或NSmT120N等体积混合共浸润本氏烟叶片，筛选与NSmWT、NSmC118Y或NSmT120N均能够产生HR反应的Sw-5bR927A突变体，最终筛选到两个能够同时与NSmWT、NSmC118Y或NSmT120N产生HR反应，且不会产生免疫自激活的Sw-5bR927A突变体。测序结果表明，两个突变体均在SD功能域33位亮氨酸(L)突变成脯氨酸(P)，即L33P；319位赖氨酸(K)突变成谷氨酸(E), 即K319E；其他功能域均未发生碱基突变。通过定点突变的方式构建植物表达载体、农杆菌瞬时浸润本氏烟叶片等手段，发现只有当三个氨基酸位点同时突变时（33位亮氨酸突变成脯氨酸，319位赖氨酸突变成谷氨酸，927位精氨酸突变成丙氨酸），Sw-5b才会与NSmWT、NSmC118Y或NSmT120N产生HR反应。此外，Sw-5b 33位亮氨酸突变成脯氨酸，319位赖氨酸突变成谷氨酸，927位精氨酸突变成谷氨酰胺后，也可以与NSmWT、NSmC118Y或NSmT120N产生HR反应（图2），且不会产生免疫自激活现象。
实施例3：Sw-5bL33P/K319E/R927A与Sw-5bL33P/K319E/R927Q转基因本氏烟对TSWV野生型以及抗性突破型株系具有系统抗病性
在Sw-5b转基因本氏烟叶片上浸润TSWV野生型及抗性突破型病毒的侵染性克隆（分别为rTSWVWT、rTSWVC118Y、rTSWVT120N），10-15天后观察本氏烟系统叶的症状并检测病毒蛋白积累量。结果表明，rTSWVWT、rTSWVC118Y和rTSWVT120N在野生型本氏烟系统叶上出现了病毒侵染之后叶片卷曲的典型症状，并能够检测到病毒蛋白的表达积累（图3与图4）。在转基因Sw-5b本氏烟上，rTSWVC118Y和rTSWVT120N能够侵染系统叶，引起叶片卷曲、萎蔫的症状；rTSWVWT无法侵染系统叶，在系统叶上观察不到TSWV引起的症状，且检测不到病毒蛋白的表达积累(图3与图4)。在转基因Sw-5bL33P/K319E/R927A与Sw-5bL33P/K319E/R927Q本氏烟上，rTSWVWT、rTSWVC118Y和rTSWVT120N均无法侵染系统叶，在系统叶上观察不到叶片卷曲、萎蔫等症状，且检测不到病毒蛋白的表达积累(图3与图4)。综上所述，Sw-5bL33P/K319E/R927A与Sw-5bL33P /K319E/R927Q对rTSWVWT、rTSWVC118Y和rTSWVT120N具有系统抗病性。
序列表
<110> 南京农业大学
<120> 抗病基因Sw-5b人工改良突变体及其应用
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Val Thr Asp Glu Glu Val Val Gly Phe Glu Asn Lys Ala Glu Glu Leu
530 535 540
Ile Asp Tyr Leu Ile Arg Gly Thr Asn Glu Leu Asp Val Val Pro Ile
545 550 555 560
Val Gly Met Gly Gly Gln Gly Lys Thr Thr Ile Ala Arg Lys Leu Tyr
565 570 575
Asn Asn Asp Ile Ile Val Ser Arg Phe Asp Val Arg Ala Trp Cys Ile
580 585 590
Ile Ser Gln Thr Tyr Asn Arg Arg Glu Leu Leu Gln Asp Ile Phe Ser
595 600 605
Gln Val Thr Gly Ser Asp Asp Asn Gly Ala Thr Val Asp Val Leu Ala
610 615 620
Asp Met Leu Arg Arg Lys Leu Met Gly Lys Arg Tyr Leu Ile Val Leu
625 630 635 640
Asp Asp Met Trp Asp Cys Met Val Trp Asp Asp Leu Arg Leu Ser Phe
645 650 655
Pro Asp Asp Gly Ile Arg Ser Arg Ile Val Val Thr Thr Arg Leu Glu
660 665 670
Glu Val Gly Lys Gln Val Lys Tyr His Thr Asp Pro Tyr Ser Leu Pro
675 680 685
Phe Leu Thr Thr Glu Glu Ser Cys Gln Leu Leu Gln Lys Lys Val Phe
690 695 700
Gln Lys Glu Asp Cys Pro Pro Glu Leu Gln Asp Val Ser Gln Ala Val
705 710 715 720
Ala Glu Lys Cys Lys Gly Leu Pro Leu Val Val Val Leu Val Ala Gly
725 730 735
Ile Ile Lys Lys Arg Lys Met Glu Glu Ser Trp Trp Asn Glu Val Lys
740 745 750
Asp Ala Leu Phe Asp Tyr Leu Asp Ser Glu Phe Glu Glu Tyr Ser Leu
755 760 765
Ala Thr Met Gln Leu Ser Phe Asp Asn Leu Pro His Cys Leu Lys Pro
770 775 780
Cys Leu Leu Tyr Met Gly Met Phe Ser Glu Asp Ala Arg Ile Pro Ala
785 790 795 800
Ser Thr Leu Ile Ser Leu Trp Ile Ala Glu Gly Phe Val Glu Asn Thr
805 810 815
Glu Ser Gly Arg Leu Met Glu Glu Glu Ala Glu Gly Tyr Leu Met Asp
820 825 830
Leu Ile Ser Ser Asn Leu Val Met Leu Ser Lys Arg Thr Tyr Lys Gly
835 840 845
Arg Val Lys Tyr Cys Gln Val His Asp Val Val His His Phe Cys Leu
850 855 860
Glu Lys Ser Arg Glu Ala Lys Phe Met Leu Ala Val Lys Gly Gln Tyr
865 870 875 880
Ile His Phe Gln Pro Ser Asp Trp Lys Gly Thr Arg Val Ser Phe Ser
885 890 895
Phe Ser Glu Glu Leu Ser Lys Phe Ala Ser Leu Val Ser Lys Thr Gln
900 905 910
Lys Pro Phe His Gln His Leu Arg Ser Leu Ile Thr Thr Asn Ala Ala
915 920 925
Lys Ser Ile Asn Asp Ile Phe Ser Cys Gln Ile Ser Glu Leu Arg Leu
930 935 940
Leu Lys Val Leu Asp Leu Ser Ser Tyr Ile Val Glu Phe Leu Ser Leu
945 950 955 960
Ala Thr Phe Lys Pro Leu Asn Gln Leu Lys Tyr Leu Ala Val Gln Ala
965 970 975
Phe Glu Phe Tyr Phe Asp Pro Gly Ser His Leu Pro His Ile Glu Thr
980 985 990
Phe Ile Val Met Asn Leu Pro Tyr Tyr Asp Ile Leu Leu Pro Val Ser
995 1000 1005
Phe Trp Glu Met Lys Lys Leu Arg His Ala His Phe Gly Lys Ala Glu
1010 1015 1020
Phe Asp Lys Gln Gly Leu Ser Glu Gly Ser Ser Lys Leu Glu Asn Leu
1025 1030 1035 1040
Arg Ile Leu Lys Asn Ile Val Gly Phe Asp Arg Val Asp Val Leu Ser
1045 1050 1055
Arg Arg Cys Pro Asn Leu Gln Gln Leu Gln Ile Thr Tyr Phe Gly Asn
1060 1065 1070
Asn Glu Glu Pro Phe Cys Pro Lys Leu Glu Asn Leu Thr Gln Leu Gln
1075 1080 1085
Gln Leu Gln Leu Ser Phe Ala Arg Pro Arg Thr Leu Ser Gly Leu Gln
1090 1095 1100
Leu Pro Ser Asn Leu Asn Lys Leu Val Leu Glu Gly Ile His Ile Gly
1105 1110 1115 1120
Cys Val Ile Pro Phe Ile Ala Gly Leu Pro Ser Leu Glu Tyr Leu Gln
1125 1130 1135
Leu His Asp Val Cys Phe Pro Gln Ser Glu Glu Trp Cys Leu Gly Asp
1140 1145 1150
Ile Thr Phe His Lys Leu Lys Leu Leu Lys Leu Val Lys Leu Asn Ile
1155 1160 1165
Ser Arg Trp Asp Val Ser Glu Glu Ser Phe Pro Leu Leu Glu Thr Leu
1170 1175 1180
Val Ile Lys Lys Cys Ile Asp Leu Glu Glu Ile Pro Leu Ser Phe Ala
1185 1190 1195 1200
Asp Ile Pro Thr Leu Glu Gln Ile Lys Leu Ile Gly Ser Trp Lys Val
1205 1210 1215
Ser Leu Glu Asp Ser Ala Val Arg Met Lys Glu Glu Ile Lys Asp Thr
1220 1225 1230
Glu Gly Cys Asp Arg Leu His Leu Val Lys Gln Arg Ser Asp
1235 1240 1245
<210> 2
<211> 1246
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Glu Asn Glu Ile Glu Glu Met Leu Glu His Leu Arg Arg Ile
1 5 10 15
Lys Ser Gly Gly Asp Leu Asp Trp Leu Asp Ile Leu Arg Ile Glu Glu
20 25 30
Pro Glu Met Val Leu Arg Val Phe Arg Thr Phe Thr Lys Tyr Asn Asp
35 40 45
Val Leu Leu Pro Asp Ser Leu Val Glu Leu Thr Lys Arg Ala Lys Leu
50 55 60
Ile Gly Glu Ile Leu His Arg Leu Phe Gly Arg Ile Pro His Lys Cys
65 70 75 80
Lys Thr Asn Leu Asn Leu Glu Arg Leu Glu Ser His Leu Leu Glu Phe
85 90 95
Phe Gln Gly Asn Thr Ala Ser Leu Ser His Asn Tyr Glu Leu Asn Asn
100 105 110
Phe Asp Leu Ser Lys Tyr Met Asp Cys Leu Glu Asn Phe Leu Asn Asp
115 120 125
Val Leu Met Met Phe Leu Gln Lys Asp Arg Phe Phe His Ser Arg Glu
130 135 140
Gln Leu Ala Lys His Arg Ser Ile Lys Glu Leu Lys Ile Val Gln Lys
145 150 155 160
Lys Ile Arg Phe Leu Lys Tyr Ile Tyr Ala Thr Glu Ile Asn Gly Tyr
165 170 175
Val Asp Tyr Glu Lys Gln Glu Cys Leu Glu Asn Arg Ile Gln Phe Met
180 185 190
Thr Asn Thr Val Gly Gln Tyr Cys Leu Ala Val Leu Asp Tyr Val Thr
195 200 205
Glu Gly Lys Leu Asn Glu Glu Asn Asp Asn Phe Ser Lys Pro Pro Tyr
210 215 220
Leu Leu Ser Leu Ile Val Leu Val Glu Leu Glu Met Lys Lys Ile Phe
225 230 235 240
His Gly Glu Val Lys Ala Ser Lys Phe Thr Gln Ser Lys Thr Phe Lys
245 250 255
Asp Lys Lys Leu Pro Lys Gly Phe Ser His His Leu His Asn Leu Leu
260 265 270
Met Tyr Leu Arg Asn Lys Lys Leu Glu Asn Phe Pro Asn Asn Ile Ala
275 280 285
Ala Gln Asn Ile Asp Val Ala Ile Glu Phe Leu Leu Val Phe Leu Asp
290 295 300
Ala Asp Val Ser Asn His Val Ile Asn Gly Asn Trp Leu Lys Glu Val
305 310 315 320
Leu Leu Lys Val Gly Ala Ile Ala Gly Asp Ile Leu Tyr Val Ile Gln
325 330 335
Lys Leu Leu Pro Arg Ser Ile Asn Lys Asp Glu Thr Ser Asn Ile Ser
340 345 350
Leu Cys Ser Ile Gln Ile Leu Glu Lys Thr Lys Asp Leu Lys Ala Gln
355 360 365
Val Glu Thr Tyr Tyr Lys Ser Leu Lys Phe Thr Pro Ser Gln Phe Pro
370 375 380
Thr Phe Gly Gly Leu Ser Phe Leu Asn Ser Leu Leu Arg Lys Leu Asn
385 390 395 400
Glu Met Ser Thr Ser Lys Ser Gly Leu Gly Phe Leu Met Lys Pro Leu
405 410 415
Leu Gly Asn Leu Glu Lys Glu Leu Ser Ser Leu Thr Ser Ile Leu Glu
420 425 430
Lys Glu Leu Ser Ser Ile Phe Arg Asp Val Val His His Glu His Asn
435 440 445
Ile Pro Lys Asp Leu Gln Arg Arg Thr Ile Asn Leu Ser Tyr Glu Ala
450 455 460
Glu Val Ala Ile Asp Ser Ile Leu Ala Gln Tyr Asn Ala Phe Leu His
465 470 475 480
Ile Phe Cys Ser Leu Pro Thr Ile Val Lys Glu Ile Lys Gln Ile Asn
485 490 495
Ala Glu Val Thr Glu Met Trp Ser Ala Asp Ile Pro Leu Asn Pro His
500 505 510
Tyr Val Ala Ala Pro Leu Lys His Leu Pro Asp Arg His Ser Asn Leu
515 520 525
Val Thr Asp Glu Glu Val Val Gly Phe Glu Asn Lys Ala Glu Glu Leu
530 535 540
Ile Asp Tyr Leu Ile Arg Gly Thr Asn Glu Leu Asp Val Val Pro Ile
545 550 555 560
Val Gly Met Gly Gly Gln Gly Lys Thr Thr Ile Ala Arg Lys Leu Tyr
565 570 575
Asn Asn Asp Ile Ile Val Ser Arg Phe Asp Val Arg Ala Trp Cys Ile
580 585 590
Ile Ser Gln Thr Tyr Asn Arg Arg Glu Leu Leu Gln Asp Ile Phe Ser
595 600 605
Gln Val Thr Gly Ser Asp Asp Asn Gly Ala Thr Val Asp Val Leu Ala
610 615 620
Asp Met Leu Arg Arg Lys Leu Met Gly Lys Arg Tyr Leu Ile Val Leu
625 630 635 640
Asp Asp Met Trp Asp Cys Met Val Trp Asp Asp Leu Arg Leu Ser Phe
645 650 655
Pro Asp Asp Gly Ile Arg Ser Arg Ile Val Val Thr Thr Arg Leu Glu
660 665 670
Glu Val Gly Lys Gln Val Lys Tyr His Thr Asp Pro Tyr Ser Leu Pro
675 680 685
Phe Leu Thr Thr Glu Glu Ser Cys Gln Leu Leu Gln Lys Lys Val Phe
690 695 700
Gln Lys Glu Asp Cys Pro Pro Glu Leu Gln Asp Val Ser Gln Ala Val
705 710 715 720
Ala Glu Lys Cys Lys Gly Leu Pro Leu Val Val Val Leu Val Ala Gly
725 730 735
Ile Ile Lys Lys Arg Lys Met Glu Glu Ser Trp Trp Asn Glu Val Lys
740 745 750
Asp Ala Leu Phe Asp Tyr Leu Asp Ser Glu Phe Glu Glu Tyr Ser Leu
755 760 765
Ala Thr Met Gln Leu Ser Phe Asp Asn Leu Pro His Cys Leu Lys Pro
770 775 780
Cys Leu Leu Tyr Met Gly Met Phe Ser Glu Asp Ala Arg Ile Pro Ala
785 790 795 800
Ser Thr Leu Ile Ser Leu Trp Ile Ala Glu Gly Phe Val Glu Asn Thr
805 810 815
Glu Ser Gly Arg Leu Met Glu Glu Glu Ala Glu Gly Tyr Leu Met Asp
820 825 830
Leu Ile Ser Ser Asn Leu Val Met Leu Ser Lys Arg Thr Tyr Lys Gly
835 840 845
Arg Val Lys Tyr Cys Gln Val His Asp Val Val His His Phe Cys Leu
850 855 860
Glu Lys Ser Arg Glu Ala Lys Phe Met Leu Ala Val Lys Gly Gln Tyr
865 870 875 880
Ile His Phe Gln Pro Ser Asp Trp Lys Gly Thr Arg Val Ser Phe Ser
885 890 895
Phe Ser Glu Glu Leu Ser Lys Phe Ala Ser Leu Val Ser Lys Thr Gln
900 905 910
Lys Pro Phe His Gln His Leu Arg Ser Leu Ile Thr Thr Asn Gln Ala
915 920 925
Lys Ser Ile Asn Asp Ile Phe Ser Cys Gln Ile Ser Glu Leu Arg Leu
930 935 940
Leu Lys Val Leu Asp Leu Ser Ser Tyr Ile Val Glu Phe Leu Ser Leu
945 950 955 960
Ala Thr Phe Lys Pro Leu Asn Gln Leu Lys Tyr Leu Ala Val Gln Ala
965 970 975
Phe Glu Phe Tyr Phe Asp Pro Gly Ser His Leu Pro His Ile Glu Thr
980 985 990
Phe Ile Val Met Asn Leu Pro Tyr Tyr Asp Ile Leu Leu Pro Val Ser
995 1000 1005
Phe Trp Glu Met Lys Lys Leu Arg His Ala His Phe Gly Lys Ala Glu
1010 1015 1020
Phe Asp Lys Gln Gly Leu Ser Glu Gly Ser Ser Lys Leu Glu Asn Leu
1025 1030 1035 1040
Arg Ile Leu Lys Asn Ile Val Gly Phe Asp Arg Val Asp Val Leu Ser
1045 1050 1055
Arg Arg Cys Pro Asn Leu Gln Gln Leu Gln Ile Thr Tyr Phe Gly Asn
1060 1065 1070
Asn Glu Glu Pro Phe Cys Pro Lys Leu Glu Asn Leu Thr Gln Leu Gln
1075 1080 1085
Gln Leu Gln Leu Ser Phe Ala Arg Pro Arg Thr Leu Ser Gly Leu Gln
1090 1095 1100
Leu Pro Ser Asn Leu Asn Lys Leu Val Leu Glu Gly Ile His Ile Gly
1105 1110 1115 1120
Cys Val Ile Pro Phe Ile Ala Gly Leu Pro Ser Leu Glu Tyr Leu Gln
1125 1130 1135
Leu His Asp Val Cys Phe Pro Gln Ser Glu Glu Trp Cys Leu Gly Asp
1140 1145 1150
Ile Thr Phe His Lys Leu Lys Leu Leu Lys Leu Val Lys Leu Asn Ile
1155 1160 1165
Ser Arg Trp Asp Val Ser Glu Glu Ser Phe Pro Leu Leu Glu Thr Leu
1170 1175 1180
Val Ile Lys Lys Cys Ile Asp Leu Glu Glu Ile Pro Leu Ser Phe Ala
1185 1190 1195 1200
Asp Ile Pro Thr Leu Glu Gln Ile Lys Leu Ile Gly Ser Trp Lys Val
1205 1210 1215
Ser Leu Glu Asp Ser Ala Val Arg Met Lys Glu Glu Ile Lys Asp Thr
1220 1225 1230
Glu Gly Cys Asp Arg Leu His Leu Val Lys Gln Arg Ser Asp
1235 1240 1245
<210> 3
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggctgaaa atgaaattga ggaaatgtta 30
<210> 4
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggggaactga gatggagtaa attttaagga 30
<210> 5
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tccttaaaat ttactccatc tcagttcccc 30
<210> 6
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
taacatttcc tcaatttcat tttcagccat 30

Claims (6)

1.抗病基因Sw-5b人工改良突变体，其特征在于，33位亮氨酸突变成脯氨酸，319位赖氨酸突变成谷氨酸，927位精氨酸突变成丙氨酸或谷氨酰胺，其余氨基酸位点保持不变，且三个氨基酸位点同时突变，所述抗病基因Sw-5b人工改良突变体氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
2.一种编码基因，其特征在于，其编码权利要求1所述突变体。
3.含有权利要求2所述编码基因的载体。
4.含有权利要求3所述载体的重组菌或重组细胞。
5.权利要求1所述抗病基因Sw-5b人工改良突变体在培育抗TSWV野生型、TSWVC118Y和TSWVT120N植物中的应用。
6.根据权利要求5所述的应用，其特征在于，所述植物为番茄。
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