CN110106269A - Bna-miR156d在控制甘蓝型油菜分枝发育中的应用 - Google Patents
Bna-miR156d在控制甘蓝型油菜分枝发育中的应用 Download PDFInfo
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Abstract
本发明涉及Bna‑miR156d在控制甘蓝型油菜分枝发育中的应用,具体涉及分离、克隆和应用一种包含Bna‑miR156d前体序列的DNA片段,该片段赋予甘蓝型油菜分枝数目增多的能力,其序列如SEQ ID NO:1所示,序列长度为788 bp,其成熟miRNA的序列如SEQ ID NO:2所示。甘蓝型油菜遗传转化的功能分析表明Bna‑miR156d参与调控甘蓝型油菜分枝的发育,这对于甘蓝型油菜遗传育种改良具有十分重要的意义。
Description
技术领域
本发明涉及甘蓝型油菜基因工程领域。具体涉及分离、克隆和通过功能验证得到一种能够增加分枝数目的甘蓝型油菜Bna-miR156d在甘蓝型油菜遗传育种改良中的应用。本发明采用RT-PCR的方法,分离到包含甘蓝型油菜Bna-miR156d前体序列的DNA片段,过量表达Bna-miR156d能够影响甘蓝型油菜的分枝发育、增加分枝数目,证实了该miRNA的功能及其应用途径。
背景技术
甘蓝型油菜(Brassica napus L.)是我国主要的油菜栽培种类,具有高产、抗逆性强、适应性广等特点。miRNA是一类内源非编码的长度约为21 nt的单链小RNA。miRNA的生物合成涉及转录、加工、修饰以及RISC结合等步骤。和蛋白编码基因类似,在miRNA基因的启动子区存在TATA box,miRNA基因通过RNA聚合酶II进行转录,得到含有5’-cap和3’-poly A尾的miRNA初始转录本(pri-miRNAs);随后在DICER-LIKE1(DCL1)酶的作用下形成miRNA的前体(pre-miRNA);DCL1和HYPONASTY LEAVES1(HYL1)以及SERRATE(SE)一起对pre-miRNA进行进一步加工,得到21 nt的miRNA;可通过序列特异性识别靶mRNA,并对靶mRNA进行切割、转录抑制或染色质修饰,从而发挥其生物学功能。植物发育过程中miRNA及其靶基因的表达量改变通常会导致植物形态和生理异常。随着高通量测序技术的发展,越来越多的研究发现miRNA参与植物生长发育的多个方面包括种子萌发、叶片形态、花的分化与发育、根的发育、营养期到生殖期的转变等(D'Ario M, Griffiths-Jones S, Kim M. Small RNAs: bigimpact on plant development. Trends Plant Sci, 2017, 22(12): 1056-1068)。此外,miRNA在植物抗病以及抵御非生物胁迫等方面也具有重要作用。
优化植物株型结构是选育高产甘蓝型油菜品种的一个重要研究方向,分枝数目是影响甘蓝型油菜产量性状的一个重要因素。利用杂种优势获得株型性状良好的杂交后代是提高油菜产量的有效途径之一,但是耗时太长。利用转基因技术获得分枝数目明显增多的甘蓝型油菜目前尚未见报道。
发明内容
本发明的目的是提供甘蓝型油菜Bna-miR156d的序列及功能,并进一步公开了该miRNA的应用。
本发明分离和应用一种包含Bna-miR156d前体序列(又称为前体miRNA,前体miRNA(pre-miRNA)是788 bp中的一部分)的DNA片段,其序列如SEQ ID NO:1所示,序列长度为788bp,其对应的miRNA成熟序列如SEQ ID NO:2所示,该成熟miRNA具有调控甘蓝型油菜分枝增多的能力。利用农杆菌介导法转化到野生型甘蓝型油菜植株中,并研究其在甘蓝型油菜体内的生物学功能,为Bna-miR156d在甘蓝型油菜遗传育种的应用提供了很好的基础。
本发明的一个目的是获得调控甘蓝型油菜株型发育的成熟miRNA,其序列如SEQID NO:2所示。
本发明的又一个目的是提供含有前述DNA片段的表达盒、重组载体、重组微生物或转基因细胞系。
本发明的又一个目的是提供(a1)或(a2)的应用:
(a1)前述的甘蓝型油菜Bna-miR156d,或前述的DNA片段,或前述的表达盒、重组载体、重组微生物或转基因细胞系,在调控甘蓝型油菜多分枝发育中的应用;
(a2)前述的甘蓝型油菜Bna-miR156d,或前述的DNA片段,或前述的表达盒、重组载体、重组微生物或转基因细胞系在甘蓝型油菜遗传育种中的应用。
本发明还提供一种增加目标植物分枝数目的转基因植物的培育方法,为提高目标植物中所述miRNA的含量,得到转基因植物;所述转基因植物的植物分枝数目多于所述目标植物。具体的,用前述的包含甘蓝型油菜Bna-miR156d前体序列的DNA片段转化目标植物,获得转基因植物;所述的目标植物为甘蓝型油菜。
更具体的,包括下述步骤:
(1)克隆包含甘蓝型油菜Bna-miR156d前体序列的DNA片段;
(2)利用电击法将带有甘蓝型油菜Bna-miR156d前体序列的质粒转化农杆菌;
(3)将带有转化质粒的农杆菌采用转基因方法转化目标植物,得到转基因植物。
携带有本发明包含Bna-miR156d前体序列的DNA片段的表达载体可通过使用Ti质粒、植物病毒载体,直接DNA转化、微注射、电击转化等常规生物技术方法导入植物细胞(Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, NewYork, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2ndEdition))。
可使用包括本发明的包含Bna-miR156d前体序列的DNA片段的表达载体转化宿主(包括甘蓝型油菜在内的多种植物),培育株型改良的植物品种。
利用DNA回收试剂盒回收上述包含Bna-miR156d前体序列的DNA片段,利用酶切连接的方法将此片段连入pMDC83骨架载体,构建该DNA片段的过量表达载体,命名为pMDC83-miR156d。
利用电转法将pMDC83-miR156d载体导入根癌农杆菌中,根癌农杆菌菌株为GV3101。通过农杆菌侵染介导的遗传转化方法将pMDC83-miR156d转化甘蓝型油菜受体材料J9712,成功获得了Bna-miR156d基因表达量相对于野生型显著提高的转基因植株,观察发现与野生型植株相比,过表达Bna-miR156d的转基因甘蓝型油菜分枝数目增多,说明Bna-miR156d能够调控植物株型。
综上所述,本发明以甘蓝型油菜为研究材料,发现了一个能够调控甘蓝型油菜分枝发育的候选miRNA,该miRNA在增加植物分枝的发育过程中起重要作用,并为甘蓝型油菜的遗传育种提供重要的理论基础。
附图说明
序列表SEQ ID NO:1是本发明分离克隆的包含Bna-miR156d前体序列的DNA片段,其长度为788 bp;
序列表SEQ ID NO:2是成熟Bna-miR156d的序列;
图1 Bna-miR156d在甘蓝型油菜各个组织中的表达情况;
图2 Bna-miR156d过量表达植株中Bna-miR156d的表达情况,WT为转基因阴性苗;OE-miR156d-6、OE-miR156d-7、OE-miR156d-9、OE-miR156d-11、OE-miR156d-14、OE-miR156d-21为Bna-miR156d转基因阳性甘蓝型油菜;
图3 Bna-miR156d过量表达转基因甘蓝型油菜的株型表型,图中:WT为野生型对照植株;OE-miR156d为过表达Bna-miR156d的转基因甘蓝型油菜。
具体实施方式
以下实施例定义了本发明,并描述了本发明在克隆包含有Bna-miR156d前体序列,以及验证Bna-miR156d功能的方法。根据以下描述和这些实施例,本领域技术人员可以确定本发明的基本特征,并在不偏离本发明精神和范围的情况下,可以对本发明做出各种改变和修改,以使其适用于不同的用途和条件。
实施例1:qRT-PCR分析Bna-miR156d在甘蓝型油菜各组织器官中的表达情况
取甘蓝型油菜各个时期不同组织样品,迅速置于液氮中速冻,并转移到70℃冰箱保存,直至RNA抽提。总RNA抽提采用TaKaRa公司的RNAiso Plus试剂盒提取。利用miRNA反转录试剂盒(miRcute Plus miRNA First-StrandcDNA Synthesis Kit)和表达量检测试剂盒SYBRGreen(miRcute Plus miRNA qPCR Detection Kit,包含反向通用引物),通过poly(A)加尾法对Bna-miR156d的表达水平进行qPCR检测。mRNA检测所用上游引物序列为:5′-TGACAGAAGAGAGTGAGCAC-3′(SEQ ID NO:3);下游引物由试剂盒提供。内参引物序列为:5′-GTCTGCCTGGGTGTCACG-3′(SEQ ID NO:4)。利用ABI 7500荧光定量PCR仪进行qPCR分析。反应程序为:95℃ 10 min,95℃ 20 s,58℃ 34 s,采集荧光信号,共40个循环;60℃ to 95℃,每1 ℃采集一次荧光信号,持续1 s。每个样品设置3次技术重复,反应结束后,用ABI7500自带的软件(7500 Software v2.0.1)进行分析及绘图,计算Bna-miR156d在甘蓝型油菜各组织器官中的相对表达量,如图1所示。
实施例2:甘蓝型油菜Bna-miR156d前体序列的分子克隆
取甘蓝型油菜品种“Darmor-bzh”三叶一心期幼苗,液氮速冻,放置-70℃冰箱中保存以备提取总RNA。总RNA抽提采用大连TaKaRa公司的RNAiso Plus试剂盒提取。甘蓝型油菜cDNA的合成按南京诺唯赞生物科技有限公司的HiScript 1st Strand cDNA Synthesis Kit说明书操作进行第一链合成。
以上述cDNA第一链为扩增模板,以设计的F:5'-ACGGAGAAGGTGAAAAGATG-3'(SEQID NO:5)和R: 5'-CTGGTCCTAACGCTTTTGAA-3'(SEQ ID NO:6)为引物,利用RT-PCR进行cDNA扩增,扩增条件为:94℃ 3 min,94℃ 15 s,59℃ 15 s,72℃ 30 s,共35个循环;72℃ 10min。PCR结束后进行电泳分析,采用诺唯赞生物科技有限公司的DNA回收试剂盒回收目的扩增片段。将扩增片段连接北京全式金生物技术有限公司的pEASY-Blunt T载体,转化大肠杆菌感受态细胞,挑取白色菌落进行菌落PCR鉴定阳性克隆,将阳性克隆送到南京擎科生物科技有限公司测序,经测序验证无误的质粒命名为pre-miR156d-T。
实施例3:Bna-miR156d过量表达载体的构建
为了更好地分析Bna-miR156d的功能,申请人将其在甘蓝型油菜中过量表达,通过观察转基因植株的表型来研究该基因的功能。
过量表达载体构建方法如下:以上述经测序验证无误的Bna-miR156d基因克隆载体质粒Bna-miR156d-T为模板,使用引物pre-miR156d F:5'-GACTAGTACGGAGAAGGTGAAAAGATG-3')(SEQ ID NO:7)(序列特异引物外加接头SpeI位点)和pre-miR156d R:5'-GGCGCGCCTTCAAAAGCGTTAGGACCAG-3'(SEQ ID NO:8)(序列特异引物外加接头AscI位点),利用PCR进行cDNA扩增,扩增条件为:94℃ 3 min,94℃ 15 s,58℃ 15s,72℃ 30 s,共35个循环;72℃ 10 min。PCR结束后进行电泳分析,采用诺唯赞生物科技有限公司的DNA回收试剂盒回收目的扩增片段。将扩增片段连接到北京全式金生物技术有限公司的pEASY-Blunt T载体中,转化大肠杆菌感受态细胞,挑取白色菌落进行菌落PCR以鉴定阳性克隆,将阳性克隆送到南京擎科生物科技有限公司测序。包含Bna-miR156d前体序列的DNA片段的克隆载体质粒经SpeI+AscI双酶切后,利用DNA回收试剂盒回收目的DNA片段,将此片段与相应酶切的pMDC83骨架载体相连构建成Bna-miR156d的过量表达载体,命名为pMDC83- miR156d。
实施例4:pMDC83-miR156d过量表达载体的甘蓝型油菜遗传转化
利用电击转化法将pMDC83-miR156d质粒导入根癌农杆菌GV3101菌株感受态细胞中。挑取单菌落接种于25 mL YEB培养基(含50 mg/L 利福平)中培养过夜,取5 mL菌液转接到100mL YEB培养基(含50 mg/L 利福平)中,培养至OD600= 0.7-0.8,将菌液在冰上放置10 min,5000 rpm 4℃离心10 min收集菌体,加入100 mL无菌双蒸水清洗两次。加入4 mL 10%甘油悬浮菌体,转移到50 mL离心管中。4℃ 5000 rpm离心10 min收集菌体,加入500 µL 10%甘油重悬菌体,转移到1.5 mL离心管中。
取50 µL感受态细胞,加入5 µL pMDC83-Bna-miR156d重组质粒,用枪头混匀后转移到0.1 cm电转化杯中。电转化参数:200 Ω,1.7 KV,2.5 F,电击后立即加入500 µL LB培养液。28℃ 220 rpm培养1 h后,取100 µL菌液涂布含有卡那霉素、庆大霉素、利福平抗性的LB培养基筛选转化子,28℃培养16 h。
甘蓝型油菜的遗传转化方法为借鉴华中农业大学作物遗传改良国家重点实验室的转化方法改进之后的方法,以甘蓝型油菜无菌幼苗的下胚轴作为外植体,利用农杆菌介导法实现外源片段在甘蓝型油菜中的遗传转化。
所用培养基配方如下:
接种培养基(M0):MURASHIGE & SKOOG MEDIUM(Duchefa Biochemie公司)+30.0 g/L蔗糖Sucrose+8 g/L 琼脂Agar(pH 5.8-pH 6.0)。
共培养培养基(M1):M0+ 18.0 g/L甘露醇Mannitol+ 1.0 mg/L 2,4-二氯苯氧乙酸2,4-D+0.3 mg/L激动素Kinetin+100 μM 乙酰丁香酮AS(pH 5.8)。
愈伤分化培养基(M2):M1+300.0 mg/L 特美汀Timentin+25 mg/L 潮霉素Hygromycin B。
生苗培养基(M3):MURASHIGE & SKOOG MEDIUM(Duchefa Biochemie公司)+10.0g/L 葡萄糖Glucose+0.25 g/L木糖Xylose+0.6 g/L 吗啉乙磺酸MES+2.0 mg/L 玉米素Zeatin+0.1 mg/L 吲哚乙酸IAA+300.0 mg/L 特美汀Timentin+25 mg/L 潮霉素Hygromycin B。
壮苗生根培养基(M4):M0+300.0 mg/L 特美汀Timentin。
MURASHIGE & SKOOG MEDIUM简称为MS培养基。
具体操作步骤如下:
(1)灭菌:
a. 首先用75%酒精浸泡甘蓝型油菜种子1 min,注意时间不能过长;
b. 接着用2%的次氯酸钠消毒20 min;
c. 最后用无菌水冲洗种子4-5遍,尽量将其清洗干净。
(2)播种:
a. 用无菌镊子将灭过菌的种子播到M0培养基上,每皿30粒;
b. 将接种的培养罐放置到培养箱中,24℃下暗培养6-7 d。
(3)摇菌:
待播种5-6 d后,将农杆菌接种于含有LB液体培养基的无菌三角瓶或离心管中,置于28℃ 180-220 rpm摇床中进行培养。
(4)制备外植体并且侵染:
a. 用无菌镊子和解剖刀切取播种后生长6-7 d的幼苗,将其下胚轴切成长度为0.8-1.0 cm的外植体段。切苗时将其下胚轴置于M1液体培养基中切取,这样切取效果更佳。切的时候要快、准,不拖拉;
b. 测量农杆菌的OD600值(LB培养基中OD600=0.3左右较好),将预先培养好的菌液以6000 rpm离心10 min,弃上清,用与菌液等体积的含有100 μM乙酰丁香酮AS的MS液体培养基将菌重悬,之后再重复一次。最终取2 mL菌液,用20 mL含有100 μM乙酰丁香酮AS的MS液体培养基稀释;
c. 将切好的外植体置于已经调整好浓度的菌液重悬液中,浸染10 min,注意侵染时间不要过长,否则会导致外植体死亡。每20 mL的菌液中浸染150-200个外植体较合适;
(5)将侵染好的外植体转移至M1培养基上,以每皿20-25个外植体为宜,置于24℃暗光下培养36-48 h;
(6)将外植体从M1转到M2培养基上,并转移至光照培养箱中培养(24℃ 光16 h/暗8 h)3周;
(7)将外植体转移到M3培养基上,每2-3周继代一次,直至出现绿芽;
(8)最后将外植体转移到M4培养基中生根,生根时间需2-4周。
实施例5:转基因甘蓝型油菜阳性植株的鉴定
采用快速法提取甘蓝型油菜基因组DNA,其步骤如下:
(1)取两片幼嫩叶片(约0.2 g),剪碎装入2 mL的离心管中,加入250 µL DNA buffer(500 mM Tris-HCl,300 mM NaCl,300 mM Sucrose,pH=7.5)和两个钢珠(直径6.7 mm),打样机50 Hz、180 s打碎叶片样品;
(2)将打碎的样品置于95℃孵育10 min;
(3)将样品取出冷却至室温,12000 rpm离心5 min;
(4)吸取50 µL上清转移至新的1.5 mL离心管中,稀释5倍后备用。
取1 µL DNA作为模板,以引物35S(5'-TCCCACTATCCTTCGCAAG-3')(SEQ ID NO:9)和pre-miR156d R(5'-GGCGCGCCTTCAAAAGCGTTAGGACCAG-3')(SEQ ID NO:8)、pre-miR156dF(5'-GACTAGTACGGAGAAGGTGAAAAGATG-3')(SEQ ID NO:7)和GFP(5'-TCAGGGTAACGGGAGAAGC -3')(SEQ ID NO:10)进行PCR扩增,扩增条件为:95℃ 5 min;95℃30 s,58℃ 30 s,72℃ 30 s,共35个循环;72℃ 10 min。以转基因甘蓝型油菜DNA为模板,能扩增出特异性目的片段,证明目的载体pMDC83-miR156d已经整合到甘蓝型油菜基因组中。
实施例6:过表达Bna-miR156d使转基因甘蓝型油菜分枝数增加
本发明采用荧光实时定量PCR的方法对部分转基因甘蓝型油菜植株中Bna-miR156d的表达进行检测,RNA的提取和Bna-miR156d表达量的检测方法见实施例1。结果显示,成功获得了Bna-miR156d表达量相对于野生型Bna-miR156d表达量显著提高的转基因植株(图2)。Bna-miR156d过表达植株中miR156d的表达水平较对照提高了3~60倍。对过表达Bna-miR156d的转基因植株进行表型分析,发现与野生型对照植株相比,过表达Bna-miR156d的转基因植株分枝数目增多(图3)。Bna-miR156d过表达植株的分枝数较对照提高了2~4倍。
本发明分离得到与株型相关的甘蓝型油菜miRNA,能够针对性获得调控植物株型的候选miRNA,对研究植物分枝机理和分离与植物株型相关miRNA具有一定的理论指导作用。本发明分离得到的株型相关基因来自植物本身,对环境影响较小。利用分离的基因进行油菜株型改良分子育种,对于培育株型改良的甘蓝型油菜新品种、提高甘蓝型油菜产量和收获指数具有非常重要的意义。
以上显示和描述了本发明的基本原理、主要特征以及本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
序列表
<110> 扬州大学
<120> Bna-miR156d在控制甘蓝型油菜分枝发育中的应用
<130> xhx2019040402
<141> 2019-04-04
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 778
<212> DNA
<213> Brassica napus L.
<400> 1
atgaggatct cagttttaga gaggaactgt acggaagtgt ccgtacattt gcttttttta 60
cttttaagat ctaatcttta tttcctttgt tctttcctgt ttggtaattt attttcctct 120
ccgcacgatt tggtcaaaac cctagatttg tgggtatcta aaaatgaata aaactttcct 180
ctcttccttt ggttataaat attctctctg gctttcttgc ttaccctaag ccctctcaga 240
tctgactcta cagcctcagg gtctccttct ttcaaacgcc ttcggtctct gattacacaa 300
tttgtgtcgg taggccatat tgagtgagtg ttttgggaca agttttagaa aaacgcaaag 360
aaactgacag aagagagtga gcacacaaag gcactttgca tgttcgatgc atttgcttct 420
cttgcgtgct cactgctcta tctgtcaggt tccggcaccg attctttttg gtccgttcac 480
gttttcttct ttgattgtgt tcccatctct ctcacttttg tgttcccatc tctctcaccc 540
actttctctc tccataggta aattatatac ggtattatat ctatactaat ttgctcttac 600
attcctagat ttgttactaa ttgcatgcga ttttgctttt tgtttattct tgttgaacgt 660
tatattctat gagatcatta atatcgaaaa tgctttaatc tatgtgttta aggcttggtt 720
aagcgagatt aacatgggac cattaacgtt aacctagttt tccttaggct ggtcctaa 778
<210> 2
<211> 20
<212> DNA
<213> Brassica napus L.
<400> 2
tgacagaaga gagtgagcac 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tgacagaaga gagtgagcac 20
<210> 4
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gtctgcctgg gtgtcacg 18
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
acggagaagg tgaaaagatg 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ctggtcctaa cgcttttgaa 20
<210> 7
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gactagtacg gagaaggtga aaagatg 27
<210> 8
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggcgcgcctt caaaagcgtt aggaccag 28
<210> 9
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tcccactatc cttcgcaag 19
<210> 10
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tcagggtaac gggagaagc 19
Claims (7)
1.包含甘蓝型油菜Bna-miR156d前体序列的DNA片段,其特征在于,所述的DNA片段的序列如SEQ ID NO:1所示。
2.甘蓝型油菜Bna-miR156d,其特征在于,其成熟序列如SEQ ID NO:2所示。
3.权利要求1所述的包含甘蓝型油菜Bna-miR156d前体序列的DNA片段或权利要求2所述的甘蓝型油菜Bna-miR156d在调控甘蓝型油菜分枝发育中的应用。
4.含有权利要求1所述的DNA片段的表达盒、重组载体、重组微生物或转基因细胞系。
5.(a1)或(a2)的应用:
(a1)权利要求2所述的甘蓝型油菜Bna-miR156d,或权利要求1所述的DNA片段,或权利要求4所述的表达盒、重组载体、重组微生物或转基因细胞系,在调控甘蓝型油菜多分枝发育中的应用;
(a2)权利要求2所述的甘蓝型油菜Bna-miR156d,或权利要求1所述的DNA片段,或权利要求4所述的表达盒、重组载体、重组微生物或转基因细胞系在甘蓝型油菜遗传育种中的应用。
6.一种增加目标植物分枝数的转基因植物的培育方法,其特征在于,用权利要求1所述的包含甘蓝型油菜Bna-miR156d前体序列的DNA片段转化目标植物,获得转基因植物;所述的目标植物为甘蓝型油菜;所述转基因植物的植物分枝数目多于所述目标植物。
7.根据权利要求6所述的一种增加目标植物分枝数的转基因植物的培育方法,其特征在于,包括下述步骤:
(1)克隆包含甘蓝型油菜Bna-miR156d前体序列的DNA片段;
(2)利用电击法将带有甘蓝型油菜Bna-miR156d前体序列的质粒转化农杆菌;
(3)将带有转化质粒的农杆菌采用转基因方法转化目标植物,得到转基因植物。
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