CN113248584A - Ralf蛋白质在促进植物对磷元素吸收中的应用 - Google Patents
Ralf蛋白质在促进植物对磷元素吸收中的应用 Download PDFInfo
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Abstract
本发明公开了一类RALF蛋白、编码RALF蛋白的核酸序列及其应用,所述RALF蛋白质为a1或a2或a3或a4或a5:其中a1由序列表中序列1所示的氨基酸组成的蛋白质;a2由序列表中序列2所示的氨基酸组成的蛋白质;a3由序列表中序列3所示的氨基酸组成的蛋白质;a4由序列表中序列4所示的氨基酸组成的蛋白质;a5由序列表中序列5所示的氨基酸组成的蛋白质;RALF23蛋白质在拟南芥中可以促进根系的发育并缓解植物磷胁迫导致的植物矮小表型并增加植物的产量,故其在提高植物对磷元素的利用效率方面具有很强的应用价值。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及到RALF蛋白质在促进植物对磷元素吸收中的应用。
背景技术
植物在生长发育过程中需要大约16种必需营养元素,其中磷元素是植物生长发育所必需的元素之一,主要因为其在能量代谢、物质代谢和光合作用等过程中起着至关重要的作用,并且是核酸、植素和卵磷脂的重要组成成分,在很大程度上决定了作物的产量和品质。磷元素由于可被利用的磷主要是以磷酸根的形式存在,而磷酸根易于与土壤中的金属离子结合形成沉淀,致使土壤中可利用磷的含量却很少能满足植物的需要。同时施入土壤的化学磷肥也极易被土壤吸附、固定和矿化,至少80%的磷肥难以被植物吸收利用。因此提高植物对磷元素的利用效率是提高产量、降低生产成本和减轻环境污染的有效措施。
浙江省农科院病毒学与生物技术研究所植物次生代谢研究团队2020年12月7日在《New Phytologist》杂志在线发表了题为“SPX4 Interacts with both PHR1 and PAP1 toRegulate Critical Steps in Phosphorus-Status Dependent AnthocyaninBiosynthesis”的研究论文,揭示了磷信号调控花青素生物合成的新机制。
花青素是一种在自然界分别广泛、抗氧化能力很强的天然植物色素,有抗衰老、增进视力、预防癌症、预防心血管疾病等功效。磷是植物生长发育所必须的大量元素,对于植物的生长和农作物增产具有重要作用。植物在缺磷情况下会通过磷信号转导通路调控一系列的生理变化来应对磷胁迫,通常,植物响应低磷或者缺磷胁迫的表型之一是在叶、茎等组织中积累花青素。
快速碱化因子Rapidalkalinizationfactor(RALF)作为一类进化保守的多肽信号分子,以基因家族形式存在,在植物生长发育中起到重要作用,其在双子叶植物拟南芥中调控花粉管生长(MurphyanddeSmet,2014),调节免疫应答(Zhangetal.,2020)。在拟南芥中至少存在35个RALF基因成员,其中RALF1、RALF22和RALF23可以调节根毛发育,生物胁迫以及非生物胁迫(Chen etal.,2016;Haruta etal.,2014;Stegmann etal.,2017;Zhao etal.,2018;Zhu etal.,2020)。
发明内容
为了缓解土壤过量施用化肥对环境的影响,并提高土壤磷元素的利用,利用生物技术手段获得促进植物幼苗根系生长并提高植物对磷等营养元素的吸收是一个行之有效的策略。本发明的目的在于提供一种生物制剂促进植物在土壤磷元素胁迫下根系的发育,提高植物对磷元素的吸收能力。
RALF蛋白质在促进植物对磷元素吸收中的应用。
RALF蛋白质为a1或a2或a3或a4或a5;
a1为SEQ ID No:1所示的氨基酸组成的蛋白质;
a2为SEQ ID No:2所示的氨基酸组成的蛋白质;
a3为SEQ ID No:3所示的氨基酸组成的蛋白质;
a4为SEQ ID No:4所示的氨基酸组成的蛋白质;
a5为SEQ ID No:5所示的氨基酸组成的蛋白质。
所述RALF蛋白质还包括:
a6)含有任一a1-a5)的融合的蛋白质;
a7)在任一a1-a5)所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质;
a8)将任一a1-a5)的蛋白质经过一个或几个氨基酸残基的取代/或缺失和/或添加产生的蛋白质;
a9)与任一a1-a5)具有70%以上结构同源性的蛋白质;
a10)与任一a1-a5)具有50%以上结构同源性且与磷等营养元素吸收相关的存在于其他植物中的同源家族蛋白。
在RALF蛋白质在促进促进植物对磷元素吸收中的应用中的植物是双子叶植物或单子叶植物;所述双子叶植物包括:茄科植物、豆科植物;所述单子叶植物包括:十字花科植物、禾本科植物、大戟科植物。
一种编码a1-a5)任一所述RALF蛋白质的生物制剂在如下(1)至(6)中至少一种中的应用:
(1)调控植物体内磷含量;
(2)调控植物对磷元素吸收速率;
(3)促进植物磷的积累和/或磷吸收;
(4)调控植物生长发育;
(5)调控植物产量和/或品质;
(6)植物育种。
所述生物制剂为包含编码a1-a5)RALF蛋白质SEQ ID No:1-5的c1-C5)核酸分子制剂,所述C1)为SEQ ID No:6所示;所述C2)为SEQ ID No:7所示;所述C3)为SEQ ID No:8所示;所述C4)为SEQ ID No:9所示;所述C5)为SEQ ID No:10所示。
所述编码RALF蛋白质核酸分子制剂为以下b1)至b7)中的任一种;
b1)含有任一c1-C5)SEQ ID No:6-10所述核酸分子的表达盒;
b2)含有任一c1-C5)SEQ ID No:6-10所述核酸分子的重组载体;
b3)含有b1)所述表达盒的重组载体;
b4)含有任一c1-C5)SEQ ID No:6-10所述核酸分子的重组微生物;
b5)含有b1)所述表达盒的重组微生物;
b6)含有b2)所述重组载体的重组微生物;
b7)含有b3)所述重组载体的重组微生物。
生物制剂应用中的植物是双子叶植物或单子叶植物;其中双子叶植物包括:茄科植物、豆科植物;其中单子叶植物包括:十字花科植物、禾本科植物、大戟科植物。
本发明公开了一类RALF蛋白、编码RALF蛋白质的核酸序列及其应用,通过转录组数据分析相关RALF基因在正常磷(1.25mM)和低磷下(10μM)的转录表达情况,发现RALF4、RALF22、RALF23、RALF33、RALF34受到低磷的诱导;以RALF23基因调节磷元素吸收为例,发现RALF23蛋白质在拟南芥中可以促进根系的发育并缓解植物磷胁迫导致的植物矮小表型并增加植物的产量,RALF23基因过表达植物具有更耐低磷胁迫的性状;枯草芽孢杆菌分泌RALF23蛋白质提高植物对低磷的响应,故其在提高植物对磷元素的利用效率方面具有很强的应用价值。
附图说明
图1为在正常磷和低磷情况下相关RALF基因的表达分析。
图2为对照组和RALF23蛋白小肽处理组表型分析。
图3为野生型(Col-0)和RALF23过表达植物在低磷条件下表型分析。
图4为枯草芽孢杆菌分泌RAL23F多肽处理组和对照组对低磷胁迫的表型分析。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1相关RALF基因在低磷条件下受到强烈诱导
首先通过转录组数据分析相关RALF基因在正常磷(1.25mM)和低磷下(10μM)的转录表达情况,发现多个RALF受到低磷的诱导,然后在正常磷(1.25mM)和低磷(10μM)下对拟南芥植物进行培养,培养7d后,进两种培养条件的植物进行RNA提取,然后采用逆转录试剂盒进行逆转录成cDNA;合成RALF1,RALF4,RALF22,RALF23,RALF33,RALF34的定量引物序列。
RALF1-QF:CTTACGATTCTCGTCGTCTTCATCATCTC
RALF1-QF:CGTGGCAGCCTGAACCATTGTCT
RALF4-QF:ACAAACCGTCGTCAACTC
RALF4-QR:ATCATTTAGCGAGCGTAC
RALF22-QF:TTCGGAGATTCGCTAGATTTCGTGAG
RALF22-QF:TCAACGCCTGCACCTAGTGATGGT
RALF23-QF:CATAGTTCGTGCACAGAGAGAGCTAAAGC
RALF23-QR:CTATTAATTATAATGTATTTATTCCATAG
RALF33-QF:TCGCCGCCGTAACCTCCCAATC
RALF33-QR:ACGCCTGTTGATCTCAGAGTCCATCTCG
RALF34-QF:CTTCTTCGCTCTAGTTTCC
RALF34-QR:CGTCGTCTTCCTCCGTAA
再用qPCR进行验证(95℃变性10s,60℃退火20s,72℃延伸10s,40个循环),结果如图1所示,发现这RALF4、RALF22、RALF23、RALF33、RALF34五个候选基因受到低磷的诱导。
以下以RALF23基因调节磷元素吸收结合附图及实施例对本发明作进一步说明,但这些具体实施方案不以任何方式限制本发明的保护范围。
实施例2在植物根部浇灌RALF23蛋白制剂缓解磷胁迫
基于原核表达蛋白技术,构建RALF23基因的原核表达载体,用体外纯化的方式合成RALF23蛋白,具体操作步骤如下:
PCR克隆:PCR反应扩增RALF23基因片段。PCR反应体系(50μL)见表1所示,PCR反应条件:95℃预变性5min;95℃变性30s,60℃退火30s,72℃延伸30s,34个循环后,72℃继续延伸10min,完成后在4℃保存。PCR产物回收:将RALF23的PCR产物进行胶回收,4℃保存。RALF23进行PCR扩增的引物为His-RALF23-F/His-RALF23-R,引物序列如下:His-RALF23-F:5'-GGCTGATATCGGATCCGCAACAAGAAGATATATTTCATATG-3'(下划线为BamHI酶切位点);
His-RALF23-R:5'-GACGGAGCTCGAATTCTGATCTTCTGCATCTTGTAATTG-3'(下划线为EcoRI酶切位点)。
表1 PCR反应体系(50μL)
原核表达载体构建:利用BamHI和EcoRI对pET32a载体进行双酶切,并回收载体片段,将所得片段与载体片段预混,利用同源重组的方法进行连接,转化感受态Top10,涂LB平板(含有50mg/LKan),37℃过夜培养后,即可获得单菌落,菌落PCR鉴定获得阳性转化子后,37℃摇菌过夜,提取质粒,并测序;所得阳性质粒转入BL21感受细胞。将携有原核表达重组质粒的BL21菌种进行IPTG诱导,28℃,4h,0.5mM IPTG。诱导后菌体经超声破碎,和Ni柱吸附纯化,纯化后蛋白储存于-20℃。
用无菌水将RALF23蛋白质稀释到终浓度为1μM,之后将缺磷胁迫7天后的拟南芥的根部浸泡含1μM RALF23的HP(高磷)或者LP(低磷)的液体培养基中,湿度30%和光/暗周期为16h/8h的气候室内共培育5天,观察表型。
结果与分析:以低磷胁迫7天的拟南芥为实验材料,通过根部浸泡RALF23蛋白质制剂后,发现RALF23蛋白质浸泡组植株恢复正常生长(图1A),且地上部分鲜重与对照组相比也显著增加(图1B)。
图2A为对照组和RALF23蛋白质处理组分别在高磷(HP)和低磷(LP)条件下的生长表型,图2B为对照组和RALF23蛋白质小肽处理组在低磷条件下培养5天后地上部分鲜重。从图2中可以看出在低磷条件下用RALF23蛋白质小肽处理后植株部分恢复正常生长且地上部分鲜重显著增加。
实施例3RALF23过表达植物耐低磷胁迫能力检测
基于农杆菌侵染技术,构建由35S启动子启动的RALF23基因过表达载体,通过农杆菌浸染技术获得35S::RALF23-GFP过表达植物,将过表达植物与野生型(Col-0)点播于1/2MS培养基上萌发培育3天,之后分别移苗至MS(正常)和LP(低磷)培养基上,在湿度30%和光/暗周期为16h/8h的气候室内共培育5天,观察表型。
图3A为野生型(Col-0)和RALF23过表达(RALF23-OE)植物在不同磷条件下生长表型。图3B为野生型(Col-0)和RALF23过表达(RALF23-OE)植物在不同磷条件下花青素积累含量统计。我们可以发现在低磷(LP)条件下,RALF23过表达(RALF23-OE)植物会积累更少的花青素,说明RALF23过表达(RALF23-OE)植物具有更耐低磷胁迫的性状。
结果与分析:我们可以发现在低磷(LP)条件下,RALF23过表达(RALF23-OE)植物会积累更少的花青素(图3),说明RALF23过表达(RALF23-OE)植物具有更耐低磷胁迫的性状。
实施例4枯草芽孢杆菌分泌RALF23蛋白质提高植物对低磷的响应
1)引物设计:从Tair网站中获得拟南芥中RALF23基因的编码序列,将RALF23基因的密码子优化为利于枯草芽孢杆菌表达的密码子,选取位于RALF23基因C端的序列为目标片段设计引物,并在上游引物F1的上游5'端加入EcoRI限制性内切酶酶切位点和载体同源臂;在下游引物R1的5'端加入HindIII限制性内切酶酶切位点和载体同源臂。
RALF23-F:5'-ACCCTCGAGGGATCCGAATTCGCAACAAGAAGATATATTTCATATG-3'(下划线为EcoRI酶切位点);
RALF23-R:5'-AGACTGCAGGTCGACAAGCTTTGATCTTCTGCATCTTGTAATTG-3'(下划线为HindIII酶切位点)
2)PCR克隆:PCR反应扩增RALF23基因片段。PCR反应体系(50μL见表1),PCR反应条件:95℃预变性5min;95℃变性30s,60℃退火30s,72℃延伸20s,34个循环后,72℃继续延伸5min,完成后在4℃保存;
3)PCR产物鉴定:将RALF23的PCR产物胶回收,与pPE-S载体连接,转化感受态大肠杆菌Top10,涂Amp抗生素的平板,采用菌落PCR进行初步筛选后,将阳性克隆送往博尚生物技术有限公司进行测序及BLAST分析;
4)获得pBE-S及pBE-S-RALF1-His、pBE-S-RALF23-His的枯草芽孢杆菌菌株:将pBE-S空载及pBE-S-RALF23-His的阳性克隆质粒转化入RIK1285感受态细胞,涂布LB平板(含有10mg/LKan)培养,37℃倒置培养16h,形成单菌落,并利用菌落PCR筛选出携带目的基因的枯草芽孢杆菌;
5)pBE-S及pBE-S-RALF23-His接种与检测:挑菌pBE-S及pBE-S-RALF23-His单克隆的枯草芽孢杆菌在5mL的LB单抗(10μg/mLkan)培养液中37℃小摇过夜。取20μl菌液到200mlLB培养基中,加入等比例的抗生素,摇菌至OD值为0.6,6000rpm离心5-10min,调菌液OD值至0.6,以人工气候室内培养低磷胁迫2-3周的拟南芥为实验材料进行灌根处理(灌根量为1mL/株);灌根后在23℃,湿度30%和光/暗周期为16h/8h的气候室内培养3-4周,观察拟南芥的表型变化。
结果与分析:以人工气候室内培养低磷胁迫2-3周的拟南芥为实验材料,通过灌根法将含有pBE-S及pBE-S-RALF23-His蛋白表达载体的枯草芽孢杆菌进行灌根处理后,发现侵染组含有pBE-S-RALF23-His蛋白表达载体的植株恢复正常生长(图4A),根系也变得比对照组的要发达(图4B),且地上部分鲜重与对照组相比也有明显的变化(图4C)。说明RALF23基因能够促进植物根系发育,提高植物磷吸收能力,从而增加植物的产量。
序列表
<110> 湖南大学
<120> RALF蛋白质在促进植物对磷元素吸收中的应用
<141> 2021-01-30
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 53
<212> PRT
<213> Arabidopsis thaliana
<400> 1
Ala Arg Gly Arg Arg Tyr Ile Gly Tyr Asp Ala Leu Lys Lys Asn Asn
1 5 10 15
Val Pro Cys Ser Arg Arg Gly Arg Ser Tyr Tyr Asp Cys Lys Lys Arg
20 25 30
Arg Arg Asn Asn Pro Tyr Arg Arg Gly Cys Ser Ala Ile Thr His Cys
35 40 45
Tyr Arg Tyr Ala Arg
50
<210> 2
<211> 49
<212> PRT
<213> Arabidopsis thaliana
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Ala Gln Lys Lys Tyr Ile Ser Tyr Gly Ala Met Arg Arg Asn Ser Val
1 5 10 15
Pro Cys Ser Arg Arg Gly Ala Ser Tyr Tyr Asn Cys Gln Arg Gly Ala
20 25 30
Gln Ala Asn Pro Tyr Ser Arg Gly Cys Ser Thr Ile Thr Arg Cys Arg
35 40 45
Arg
<210> 3
<211> 50
<212> PRT
<213> Arabidopsis thaliana
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Ala Thr Arg Arg Tyr Ile Ser Tyr Gly Ala Leu Arg Arg Asn Thr Ile
1 5 10 15
Pro Cys Ser Arg Arg Gly Ala Ser Tyr Tyr Asn Cys Arg Arg Gly Ala
20 25 30
Gln Ala Asn Pro Tyr Ser Arg Gly Cys Ser Ala Ile Thr Arg Cys Arg
35 40 45
Arg Ser
50
<210> 4
<211> 49
<212> PRT
<213> Arabidopsis thaliana
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Ala Thr Thr Lys Tyr Ile Ser Tyr Gly Ala Leu Arg Arg Asn Thr Val
1 5 10 15
Pro Cys Ser Arg Arg Gly Ala Ser Tyr Tyr Asn Cys Arg Arg Gly Ala
20 25 30
Gln Ala Asn Pro Tyr Ser Arg Gly Cys Ser Ala Ile Thr Arg Cys Arg
35 40 45
Arg
<210> 5
<211> 56
<212> PRT
<213> Arabidopsis thaliana
<400> 5
Tyr Trp Arg Arg Thr Lys Tyr Tyr Ile Ser Tyr Gly Ala Leu Ser Ala
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Asn Arg Val Pro Cys Pro Pro Arg Ser Gly Arg Ser Tyr Tyr Thr His
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Asn Cys Phe Arg Ala Arg Gly Pro Val His Pro Tyr Ser Arg Gly Cys
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Ser Ser Ile Thr Arg Cys Arg Arg
50 55
<210> 6
<211> 159
<212> DNA
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gctagaggca gaagatatat tggctatgat gcactgaaaa agaataatgt tccgtgcagc 60
agaagaggca gatcatatta tgattgcaaa aaacgcagaa gaaataatcc gtatagaaga 120
ggctgctcag caattacaca ttgctataga tatgcaaga 159
<210> 7
<211> 147
<212> DNA
<213> Arabidopsis thaliana
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gcacagaaaa aatatattag ctatggcgca atgagaagaa atagcgttcc gtgcagccgc 60
agaggcgcga gctattataa ttgccaaaga ggcgcgcaag caaatccgta tagccgcggc 120
tgcagcacaa ttacacgctg cagaaga 147
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<213> Arabidopsis thaliana
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gctacgagga ggtacatcag ctatggtgcg ctgaggagaa acacaattcc gtgctcacgt 60
cgcggcgcat cttactacaa ttgtcgacgt ggcgctcagg ccaatcctta ctctcgtggc 120
tgcagcgcca tcactcgctg ccggcgctca tga 153
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<213> Arabidopsis thaliana
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gcgacaacca aatatatcag ctatggagca ctgcggagaa atacagttcc gtgcagcaga 60
agaggcgcaa gctattataa ttgcagaaga ggagcgcagg cgaatccgta tagcagaggc 120
tgcagcgcga ttacacgctg cagaaga 147
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<212> DNA
<213> Arabidopsis thaliana
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tattggagaa gaacaaaata ttatattagc tatggcgcgc tgagcgcgaa tagagttccg 60
tgcccgccga gaagcggcag aagctattat acacataatt gctttagagc gagaggcccg 120
gttcatccgt atagccgcgg ctgcagcagc attacaagat gccgcaga 168
Claims (8)
1.RALF蛋白质在促进植物对磷元素吸收中的应用。
2.根据权利要求1所述的应用,其特征在于,所述RALF蛋白质为a1或a2或a3或a4或a5;
a1为SEQ ID No:1所示的氨基酸组成的蛋白质;
a2为SEQ ID No:2所示的氨基酸组成的蛋白质;
a3为SEQ ID No:3所示的氨基酸组成的蛋白质;
a4为SEQ ID No:4所示的氨基酸组成的蛋白质;
a5为SEQ ID No:5所示的氨基酸组成的蛋白质。
3.根据权利要求2所述的应用,其特征在于,所述RALF蛋白质还包括:
a6)含有任一a1-a5)的融合的蛋白质;
a7)在任一a1-a5)所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质;
a8)将任一a1-a5)的蛋白质经过一个或几个氨基酸残基的取代/或缺失和/或添加产生的蛋白质;
a9)与任一a1-a5)具有70%以上结构同源性的蛋白质;
a10)与任一a1-a5)具有50%以上结构同源性且与磷等营养元素吸收相关的存在于其他植物中的同源家族蛋白。
4.根据权利要求1-3任一所述的应用,其特征在于,所述植物是双子叶植物或单子叶植物;所述双子叶植物包括:茄科植物、豆科植物;所述单子叶植物包括:十字花科植物、禾本科植物、大戟科植物。
5.一种与权利要求1所述RALF蛋白质相关的生物制剂在如下(1)至(6)中至少一种中的应用:
(1)调控植物体内磷含量;
(2)调控植物对磷元素吸收速率;
(3)促进植物磷的积累和/或磷吸收;
(4)调控植物生长发育;
(5)调控植物产量和/或品质;
(6)植物育种。
6.根据权利要求5所述的应用,其特征在于,所述生物制剂为包含编码a1-a5)RALF蛋白质SEQ ID No:1-5的c1-C5)核酸分子制剂;
所述C1)为SEQ ID No:6所示;
所述C2)为SEQ ID No:7所示;
所述C3)为SEQ ID No:8所示;
所述C4)为SEQ ID No:9所示;
所述C5)为SEQ ID No:10所示。
7.根据权利要求6所述的应用,其特征在于,所述编码a1-a5)RALF蛋白质核酸分子制剂为以下b1)至b7)中的任一种;
b1)含有任一c1-C5)SEQ ID No:6-10所述核酸分子的表达盒;
b2)含有任一c1-C5)SEQ ID No:6-10所述核酸分子的重组载体;
b3)含有b1)所述表达盒的重组载体;
b4)含有任一c1-C5)SEQ ID No:6-10所述核酸分子的重组微生物;
b5)含有b1)所述表达盒的重组微生物;
b6)含有b2)所述重组载体的重组微生物;
b7)含有b3)所述重组载体的重组微生物。
8.根据权利要求5-7任一所述的应用,其特征在于,所述植物是双子叶植物或单子叶植物;所述双子叶植物包括:茄科植物、豆科植物;所述单子叶植物包括:十字花科植物、禾本科植物、大戟科植物。
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