CN102965382B - 一种与致病力相关的灰葡萄孢新基因及其应用 - Google Patents
一种与致病力相关的灰葡萄孢新基因及其应用 Download PDFInfo
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- CN102965382B CN102965382B CN201210519803.9A CN201210519803A CN102965382B CN 102965382 B CN102965382 B CN 102965382B CN 201210519803 A CN201210519803 A CN 201210519803A CN 102965382 B CN102965382 B CN 102965382B
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Abstract
本发明涉及一种灰葡萄孢基因,命名为Bcdp1,该基因含有3个外显子和2个内含子,DNA全长1608bp,cDNA全长597bp,编码198个氨基酸。本发明还涉及含有该基因的表达载体和宿主以及该基因在植物病害(特别是灰霉病)防治中的应用以及作为用于植物病害防治的药物的靶标的应用。
Description
技术领域
本发明涉及植物病害防治领域,具体地,本发明涉及一种与灰葡萄孢致病力相关Bcdp1基因。本发明还涉及该基因在植物病害防治中的应用以及作为用于植物病害防治的药物的靶标的应用。
背景技术
灰葡萄孢(Botrytis cinerea)属半知菌亚门、丝孢纲、丝孢目、葡萄孢属,它是最早被研究的真菌之一。该菌菌丝有隔膜,有性繁殖的方式主要为异宗配合,自然条件下几乎不产生子囊孢子。分生孢子梗多为褐色、直立、有分隔大小为960~1
200 µm×16~22 µm,顶端具不规则树状分支分枝,顶端稍膨大,呈棒头状,其上着生大量的分生孢子,分生孢子聚集成葡萄穗状。分生孢子呈圆形或卵形,颜色为淡灰色、灰白色或无色,其大小为8~14
µm×6~9 µm。灰葡萄孢在培养基上培养1周后,会产生菌核,菌核呈灰黑色或黑色,形状呈片状、圆形、不规则形,大小约小2~4 mm×1~3 mm;剖开菌核观察其组织,外部为疏丝组织,内部为拟薄璧组织且有大量分生孢子。
灰霉病由灰葡萄孢侵染所致,属真菌病害,它能危害番茄、黄瓜、草莓、辣椒、葡萄等多种蔬菜、果树和观赏植物,目前已发现的寄主植物至少有235种,且花、果、叶、茎均可发病。寄主植株从苗期到挂果期都可以发病,叶、茎、花、果实均可感染病害。感病后,叶部症状表现有“V”形病斑、不规则病斑、叶柄折断等症状。茎部症状表现为茎腐烂、茎基腐烂。花部症状表现为花腐烂和凋萎。果实症状表现为腐烂和果实脱落。病害发生、蔓延与湿度和温度有十分密切的关系。近年来,灰霉病的发生日益严重,给国民经济带来严重影响。目前对于灰霉病的防治仍以化学防治、为主,但由于灰葡萄孢很容易产生抗药性,而且大量使用化学药剂所造成的农药残留、环境污染等诸多问题日益严重,如何利用新的环保的方法有效防治病害成为当前急需解决的问题。
发明内容
本发明的目的在于提供一种新的灰葡萄孢致病基因,所述基因在灰葡萄孢致病过程中起重要作用,且可使用该基因在植物病害防治的靶标药物中的应用。
本发明前期借助农杆菌AGL-1(其中带有双元载体质粒pBHtl)构建了灰葡萄孢ATMT突变体库。通过筛选该突变体库,获得了大量的分生孢子形态、产孢量、菌核的有无及致病性方面发生明显变化的突变体。
进一步本发明利用离体果实接种法,从灰葡萄孢ATMT突变体库中筛选获得了一株致病力丧失的突变体Bct89。对突变体的表型分析,发现该突变体Bct89的生长速度缓慢、不产生菌核和分生孢子;显微观察发现,该菌株菌丝隔变短、变细,且颜色较野生型明显变浅。
进一步本发明利用TAIL-PCR技术,获得了致病力丧失突变体Bct89的侧翼序列。生物信息学方法确定了突变基因为BC1G_10703.1(Botrytis cinerea
pathogenic deficient related,Bcdp1),如SEQ No.1所示,且T-DNA的插入位点位于该基因的第3个外显子上。生物信息学分析发现该基因为一未知功能新基因。利用PCR技术,进一步确定了T-DNA插入到了Bcdp1基因的第3个外显子上;通过Southern
blotting技术,确定了T-DNA是以单拷贝的形式插入到突变体Bct89的基因组中;利用RT-PCR技术,进一步确定了突变体Bct89中Bcdp1基因被突变。推测Bcdp1基因参与灰葡萄孢的致病性、分生孢子发育和菌核的形成。
进一步本发明对细胞壁降解酶及毒素活性分析,发现突变体Bct89的细胞壁降解酶类纤维素酶(Cx)、多聚半乳糖醛酸酶(PG)、果胶甲基半乳糖醛酸酶(PMG)、多聚半乳糖醛酸反式消除酶(PGTE)和果胶甲基反式消除酶(PMTE)的酶活性较野生型明显降低,而毒素活性较与野生型相比差异不显著。利用RT-PCR技术,检测突变体Bct89中抗病相关基因的表达情况,发现初步Bcdp1基因突变影响胞壁降解酶基因、产毒基因、信号途径相关基因的表达。推测Bcdp1基因是通过影响灰葡萄孢的细胞壁降解酶、毒素活性及致病相关基因的表达等因素影响其致病力。
更进一步本发明成功构建了Bcdp1基因的同源重组载体,利用PEG介导的原生质体转化技术转化灰葡萄孢野生型菌株,利用潮霉素B筛选获得了10株转化子。通过PCR、RT-PCR和Southern blotting技术验证转化子,试验结果表明本课题组已经成功获得了Bcdp1基因的T-DNA插入突变体和敲除突变体Δ Bcdp1。并采用活体和离体果实、叶片接种的方法对突变体进行致病力测定,发现Δ Bcdp1和Bct89突变体均丧失了致病能力,明确了本发明所述Bcdp1基因为灰葡萄孢致病相关基因。
进一步本发明提供了所述基因作为植物病害防治的药物的靶标的应用,所述植物病害是由灰葡萄孢导致的灰霉病。发明还提供了所述基因作为一种治疗由灰葡萄孢导致的植物灰霉病的方法,包含阻断或抑制灰葡萄孢中Bcdp1基因的表达。此外,本发明还提供了所述基因在作为阻断或抑制基因的表达的药剂中的应用。
附图说明
图1 灰葡萄孢ATMT突变体库的构建图
图2 转化子的PCR验证图
1-10,不同ATMT转化子;W,对照;M,DL2000 maker。
图3 转化子的southern blot 验证图
1-7,不同ATMT转化子;W,对照;M,Southern maker。
图4 致病力丧失菌株BCt89的筛选及显微观察图
图5致病力丧失菌株BCt89遗传稳定性观察图
T5,5代;T10,第10代;T15,第15代;M-N,BCt89针刺接种;WT-N,野生型针刺接种;M-C,BCt89未针刺接种;WT-C,BCt89未针刺接种。
图6 致病力丧失菌株BCt89的表型观察图
图7 突变体BCt89的突变基因的确定
A,基因结构图;B,PCR验证 1引物P1&P2
2 引物P2&P3 3 引物P3&P4;
C,southern验证 1,WT 2,BCt89
Hind III酶切 3,BCt89
EcoRI酶切
图8突变体BCt89的致病因子比较分析(细胞壁降解酶酶活力)
PMG,果胶甲基半乳糖醛酸酶; PG,多聚半乳糖醛酸酶;Cx纤维素酶 ; PMTE,果胶甲基反式消除酶; PGTE,多聚半乳糖醛酸反式消除酶
图9突变体BCt89的致病因子比较分析(次生代谢产物)
图10 Bcdp1基因的同源重组载体的构建及转化子的PCR和RT-PCR鉴定
A,B Δ Bcdp1突变体PCR验证;C,敲除载体构建示意图 D,Southern 验证敲除转化子WT,野生型, 1-4为敲除转化子;E,RT-PCR验证敲除转化子
图11Δ Bcdp1突变体和ATMT突变株Bct89的致病力观察图
注:“-N”标注为针刺接种,其余为未针刺对照。
图12Δ Bcdp1突变体和ATMT突变株Bct89的组织病理学观察图
具体实施方式
下文将参考实施例详细描述本发明,所述实施例仅是意图举例说明本发明,而不是意图限制本发明的范围。本发明的范围由后附的权利要求具体限定。
实施例
1 Bcdp1
基因
T-DNA
插入突变体的获得
本发明所涉及的突变体是通过根癌农杆菌介导的转化法(ATMT)获得的。
1.1农杆菌的培养。 挑取农杆菌单菌落于LB液体培养基(含50 μg/mL卡那霉素及50 μg/mL利福平)中,28℃120 rpm下摇培48 h,取1 mL菌液于4℃10000 rpm下离心1 min,弃上清。用诱导培养基(IM)洗2次,并用IM培养基稀释至OD600≈0.15,加入终浓度为200~400 μM的AS,然后28℃180 rpm下活化约7 h。
1.2 灰葡萄孢分生孢子的收集。 用10 mL灭菌蒸馏水从培养大约7 d的PDA平板上洗下灰葡萄孢的分生孢子,两层纱布过滤后用血球计数板计数,并用无菌水稀释孢子浓度为105个/mL。
1.3农杆菌与灰葡萄孢孢子共培养及转化子筛选。 取100 μL培养好的农杆菌AGL-1(含载体)菌液和100 μL稀释好的灰葡萄孢分生孢子悬浮液混合,混合液(200 μL)均匀涂于CM平板的玻璃纸上,20℃培养48 h。然后将玻璃纸膜转移到涂于含有潮霉素B(50 μg/mL)、羧苄青霉素和头孢噻肟钠(各200 μg/mL)的PDA培养基上,将平皿置于20℃下培养到转化子出现。将转化子接种到含100 μg/mL潮霉素的PDA平板上再次鉴定。
1.4转化子的纯化及保存。 按上述方法获得的转化子转接到试管PDA斜面培养基上在黑暗下培养7~10天后置于4℃保存。
实施例
2
致病力丧失突变体的筛选及稳定性检测
利用果实离体接种法对灰葡萄孢T-DNA插入突变体库进行筛选。先选取大小一致的新鲜番茄用75%酒精擦洗果实进行表面消毒。同时,将复壮后的灰葡萄孢野生型BC22、转化子培养3 d,分别用打孔器从菌落边缘打取直径5 mm的菌盘,对称接种于番茄果实(无伤口),将接种后的番茄果实放入保湿缸中,6 d后调查病情。根据病斑面积大小评定菌株致病力差异。将筛选得到的灰葡萄孢致病力丧失的突变体Bct89在PDA培养基中继代培养15代(含有潮霉素100 μg/mL),并将各代进行致病力测定,观察其致病稳定性,方法同上。
实施例
3
突变体的生物学研究
将灰葡萄孢野生型菌株BC22、突变体菌株Bct89进行复壮,复壮后分别将其接种于PDA培养基上,培养3 d后,分别于菌落边缘打孔直径9 mm的野生型、突变体菌盘,20℃培养10 d,观察菌落形态、颜色,测量生长速度,产孢量等。
实施例
4
分析
T-DNA
在突变体菌株
Bct89
基因组中的插入部位
利用TAIL-PCR技术获得T-DNA在突变体菌株Bct89基因组中的插入部位。T-DNA左右边界嵌套引物、随机引物的合成及PCR程序参照Mullins等的方法。
实施例
5
突变体的
PCR
、
Southern blotting
鉴定
4.1转化子PCR鉴定
根据T-DNA中潮霉素磷酸转移酶基因设计引物,利用PCR技术对灰葡萄孢转化子DNA进行扩增。试验所需引物见表1。
表1 试验所需引物
Name | Used | Sequences (5′→3′) |
89F(P1) | Target gene | 5'-GACGGGCTGATGGACTAT-3' |
89R(P3) | Target gene | 5'-TGTGATGAGATGCAGAAGAC-3' |
HPH R(P2) | HPH; probe | 5'-CGCCCAAGCTGCATCATCGAA-3' |
HPH F(P4) | HPH; probe | 5'-CGACAGCGTCTCCGACCTGA-3' |
RTF(P5) | RT-PCR | 5'-CCTTGGCAACAAAGACCT-3' |
RTR(P6) | RT-PCR | 5'-GAATGCCGAATGTGATG-3' |
TubulinF | 内参 | 5'-ACTGGGCTAAGGGTCATT-3' |
TubulinR | 内参 | 5'-TCTCCGTAAGATGGGTTG-3' |
LB1 | TAIL-PCR | 5'-GGGTTCCTATAGGGTTTCGCTCATG-3' |
LB2 | TAIL-PCR | 5'-CATGTGTTGAGCATATAAGAAACCCT-3' |
LB3 | TAIL-PCR | 5'-GAATTAATTCGGCGTTAATTCAGT-3' |
RB1 | TAIL-PCR | 5'-GGCACTGGCCGTCGTTTTACAAC-3' |
RB2 | TAIL-PCR | 5'-AACGTCGTGACTGGGAAAACCCT-3' |
RB3 | TAIL-PCR | 5'-CCCTTCCCAACAGTTGCGCA-3' |
AD1 | TAIL-PCR | 5'-TGAGNAGTANCAGAGA-3' |
AD2 | TAIL-PCR | 5'-AGTGNAGAANCAAAGG-3' |
AD3 | TAIL-PCR | 5'-CATCGNCNGANACGAA-3' |
AD4 | TAIL-PCR | 5'-TCGTNCGNACNTAGGA-3' |
AD5 | TAIL-PCR | 5'-NTCGA(G/C)T(A/T)T(G/C)G(A/T)GTT-3' |
AD6 | TAIL-PCR | 5'-NGTCGA(G/C)(A/T)GANA(A/T)GAA-3' |
AD7 | TAIL-PCR | 5'-TG(A/T)GNAG(A/T)ANCA(G/C)AGA-3' |
AD8 | TAIL-PCR | 5'-AG(A/T)GNAG(A/T)ANCA(A/T)AGG-3' |
AD9 | TAIL-PCR | 5'-TCGTTCCGCA-3' |
扩增体系:ddH2O 17.0 μL,10×PCR Buffer 2.5 μL,dNTPs(2.5 μmol/L)2.0 μL,primer hph-S(10 μmol/L)0.5 μL,primer hph-AS(10 μmol/L)0.5 μL,模板(DNA) 1.0 μL,Taq DNA 聚合酶(5 U/μL)0.5 μL。扩增程序:95℃ 5 min;94℃ 30 s;55℃ 30 s;72℃ 1 min;Go to step 2 35 cycles;72℃ 10 min;10℃ pause。
4.2
Southern bloting分析及半定量RT-PCR分析
采用常规Southern bloting技术方法和半定量RT-PCR对突变体进行分析。
实施例
6
敲除突变体的的创制
采用常规方法构建敲除载体,并按照实施例1构建敲出突变体。
实施例
7
突变体细胞壁降解酶及毒素活性分析
灰葡萄孢存在多种致病因子参与侵染寄主植物组织的过程,这些致病因子中,有的参与信号识别及信号传导,有的破坏、降解植物细胞壁(如细胞壁降解酶类等),还有产生毒素,以及抵抗寄主防卫反应的酶类及小分子物质等,这些致病因子共同作用,相互协作。其中细胞壁降解酶类和毒素类是主要的致病因子。
5.3.1
细胞壁降解酶活性测定
(1)胞壁降解酶粗酶液的提取。灰葡萄病菌在25℃下PDA平板上培养4 d,用内径5.0 mm打孔器在PDA培养基上生长的病菌菌落边缘打孔,挑取5块菌盘移至50 mL改良的Marcus培养液中28℃下培养,6 d后过滤除去菌丝,上清液经4℃ 10 000 rpm离心15 min,反复离心2次。向酶液中缓慢加入硫酸铵搅拌,至60%的饱和度(25℃),4℃下静置5 h后10 000 rpm 4℃下离心20
min,弃上清液,用20 mL 50 mmol/L粗醋-醋酸钠缓冲液(pH5.0)溶解沉淀。在同样的缓冲液中透析48 h,每12 h换一次透析液,纯化的酶在−18℃保存备用。
(2)胞壁降解酶粗酶液活性测定。利用分光光度计在540
nm处测定反应液消光值,根据酶反应所释放的还原糖计算Cx、PG和PMG的活性。
①Cx活性:取1.0 mL粗酶液,加入1.0 mL柠檬酸-柠檬酸钠缓冲液和1.0 mL含1.0%的CMC缓冲液,50℃酶解30 min后,立即加入1.5 mL DNS终止反应,沸水浴10 min,取出流水冷却,加入8.0 mL蒸馏水在540 nm处测定其酶活,对照在终止反应后加入底物。酶活单位为50℃下每小时催化底物释放1 μmol还原糖所需酶量。
②PG、PMG活性:取0.5 mL粗酶液,加入0.5 mL PG和PMG的反应底物和1.0 mL的蒸馏水。50℃酶解1 h后,立即加入DNS 2.5 mL终止反应,沸水浴10 min,取出流水冷却,加入8.0 mL的蒸馏水在540 nm处测定其酶活,对照在终止反应后加入底物。酶活单位为50℃下每小时催化底物释放1 μg还原糖所需酶量。
③PGTE、PMTE活性:按Hoffman方法在232 nm处测定反应混合液的消光值,计算PGTE和PMTE的活性。反应混合液为1.0 mL酶液,1.0 mL Gly-NaOH缓冲液,1.0 mL底物(1%多聚半乳糖醛酸或1%果胶,pH9.0),1.0 mL 3 mM CaCl2。上述混合物在30℃水浴锅中反应10 min,在232 nm处测定反应前后的OD值。30℃下每分钟催化底物释放1 μmol不饱和醛酸所需酶量为一个酶活单位。
所有酶活测定均重复3次。酶蛋白浓度按Bradford方法,用考马斯亮兰G-250显色,在595 nm下比色测定,用牛血清作标准曲线。
5.3.2
毒素的提取及活性测定
在250 mL三角瓶内装入150 mL改良Fries培养基,每瓶接入5片菌盘(Ф=10 mm),置25℃恒温培养箱内黑暗条件下静止培养10 d,无菌条件下用两层纱布过滤,然后用定性滤纸过滤,所得液体即为培养滤液。
将培养滤液在无菌条件下定量移入冷冻干燥瓶,低温冰箱内冷冻,之后在低温冷冻干燥机上浓缩至原体积的1/6,然后在浓缩液中加等量的甲醇,离心去沉淀,旋转蒸发去甲醇,对水溶液进行萃取,即,在水溶液中加等体积的乙酸乙酯,磁力搅拌器搅拌均匀,在分液漏斗内静置24 h,收集乙酸乙酯相,完成第一次萃取,之后在水相中加等体积的乙酸乙酯再次萃取,共萃取3次,乙酸乙酯相留待制备毒素。将收集的乙酸乙酯相旋转蒸发去掉溶剂,加定量甲醇定容用于生物测定。取10 μL滴于受损的番茄果实表面,分别以甲醇、水和野生型作为对照,观测其毒素活力。
致病力测定按照实施例2的方案进行。
<110> 河北农业大学
<120> 一种与致病力相关的灰葡萄孢新基因及其应用
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1608
<212> DNA
<213> 灰葡萄孢基因组
<400> 1
atggctgaga ggttaccgac tgcgtggttc agcagggtgt gtgcagaagg
aatgcgtttt 60
agattggaga taaaagctga aaaagtaagc ctagtgtata tgctaagcag
gaacttgttc 120
gtcaacgaga gaatcaatca atgaacgtgt ccatatgtgg tctggtctgg
tctggtctgg 180
tgtaggtact cggcgagtgc gaacaggtca agaacctaag ggttttaagt
ttcccgtccc 240
gccccatcgc tttcctccac ctccacctcc acctccacac ctatacatgt
ttcatttcca 300
aacacaacga actcattttc gtagcgaaat atctctgtcg actaggccca
ccacctgatc 360
ggaccttcat gtacagaagc aaaacatttc tcgcgcaaac tcagaaccgg
aaatcggagc 420
tgaacctgac ctaggtaatg ttacacgaca actagggaca ttccggggtg
ccaagactcc 480
tgatgtacaa acccgtgaat cattgatgta ctttgcacaa acggacgggc
tgatggacta 540
tcaggtcgtg atcgatagtg acaagaagaa aaatatatca cattattacg
gaggattaga 600
acgaagcctg tacgcgtctt gacacgggtg gtcttgccgc ttgtgtaaca
ggttttgtat 660
tacggaccta acatgtcatt gatgcatgtg agtatgtatt tttcttcttt
cttagactcc 720
ttggagtgca gtttggagtg catacatgcc ggtacataca tcgatattgt
gtctttcttt 780
tttttccttc tctgcacata atacatatgc gctgcagccg catcacagcg
attcaaaacg 840
tggcagccca atatctaacc aaacccaagc cttatctaag aacgccatct
ctaacaaaca 900
gctgattgtg gaaccagagg ttcactgact caaaaccgtc tctgcccctt
caacatctgc 960
gaagaatatg attcgggata ttgaggaatg ctcgaatagc ttgaattgct
ttaatgatta 1020
ataattatta aagtttttat aaattaactg cattagtttt tcggagagat
ttgtgatgtt 1080
cgtttaatag gtgggggtgg ctctgattat catggtggga atgtgagaag
ggcgtactcc 1140
atagcggaat gagaagtgtc gctgggttca tgggaggaga atctatatac
agggaagagt 1200
ttgtcgcgga agagcaaatg gagaaggatt atcgaggagt gaagttggga
tgaagggtta 1260
cacaagattt ctcgtcttta tacaatcact gatgttattg aagaattttg aagtaataac
1320
acgagtagta tgatgaaaat catgtgtgaa aataccttgg caacaaagac
ctcgatgaag 1380
gggttcaatc tattccacat tgaaaccaat cagcgacttg caaatttaca
tcgattcaat 1440
cattcatttc tcaaaaactt ggctcagcat tcacgtcaat cgaacgttga
cactaacttg 1500
aatcctttca gagcaatgac aagtcttctg catctcatca cattcggcat
tcggtactca 1560
ctgaaaattt cgcttatact cccccattcg agtattcgtc
gtgaataa
1608
<210> 2
<211> 198
<212> PRT
<213> 灰葡萄孢
<400> 2
Met Ala Glu Arg Leu Pro Thr Ala Trp Phe Ser Arg Val Cys Ala Glu
1
5
10
15
Gly Met Arg Phe Arg Leu Glu Ile Lys Ala Glu Lys Lys Gln Asn Ile
20
25
30
Ser Arg Ala Asn Ser Glu Pro Glu Ile Gly Ala Glu Pro Asp Leu Gly
35
40
45
Asn Val Thr Arg Gln Leu Gly Thr Phe Arg Gly Ala Lys Thr Pro Asp
50
55
60
Val Gln Thr Arg Glu Ser Leu Met Tyr Phe Ala Gln Thr Asp Gly Leu
65
70
75
80
Met Asp Tyr Gln Val Val Ile Asp Ser Asp Lys Lys Lys Asn Ile Ser
85
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95
His Tyr Tyr Gly Gly Leu Glu Arg Ser Leu Met Met Lys Ile Met Cys
100
105
110
Glu Asn Thr Leu Ala Thr Lys Thr Ser Met Lys Gly Phe Asn Leu Phe
115
120
125
His Ile Glu Thr Asn Gln Arg Leu Ala Asn Leu His Arg Phe Asn His
130
135
140
Ser Phe Leu Lys Asn Leu Ala Gln His Ser Arg Gln Ser Asn Val Asp
145
150
155
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Thr Asn Leu Asn Pro Phe Arg Ala Met Thr Ser Leu Leu His Leu Ile
165
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175
Thr Phe Gly Ile Arg Tyr Ser Leu Lys Ile Ser Leu Ile Leu Pro His
180
185
190
Ser Ser Ile Arg Arg Glu
195
Claims (6)
1.一种来自灰葡萄孢(Botrytis cinerea)的基因Bcdp1,其核苷酸序列如SEQ ID No.
1所示。
2.权利要求书1所述的基因编码的多肽,其氨基酸序列如SEQ ID No.2所示。
3.权利要求l所述的基因在防治由灰葡萄孢导致的灰霉病中的应用,其中所述应用为抑制或阻断该基因在灰葡萄孢中的表达。
4.权利要求l所述的基因作为用于植物病害防治的药物的靶标的应用,所述植物病害是由灰葡萄孢导致的灰霉病。
5.一种治疗由灰葡萄孢导致的植物灰霉病的方法,包含阻断或抑制灰葡萄孢中Bcdp1基因的表达,其中所述Bcdp1基因的核苷酸序列如SEQ ID No.1所示。
6.阻断或抑制权利要求l所述基因的表达的药剂在制备药物中的应用,所述药剂是权利要求l所述基因的反义RNA或siRNA,所述药物用于控制由灰葡萄孢导致的植物灰霉病。
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