CN116410989A - 一种病毒诱导的三七pds基因沉默体系及应用 - Google Patents
一种病毒诱导的三七pds基因沉默体系及应用 Download PDFInfo
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Abstract
本发明涉及一种病毒诱导的三七PDS基因沉默体系及应用,本发明所述的三七PnPDS基因具有如SEQ ID NO.1所示的序列,沉默体系携带如SEQ ID NO.2所示的序列。通过构建含有PnPDS特异片段的pTRV2病毒沉默载体,利用农杆菌介导法比较了不同浸染方式对三七不同时期幼苗浸染后的效果,发现真空渗透处理三七子叶期幼苗能有效诱导三七PnPDS基因沉默,显著降低了三七叶片中PnPDS基因的表达水平,导致三七叶片呈现漂白化表型。本发明首次成功构建了三七PnPDS基因的VIGS体系,该体系的建立为研究三七基因功能提供了有效的技术手段。
Description
技术领域
本发明属于植物基因功能研究技术领域,尤其是涉及一种病毒诱导的三七PDS基因沉默体系和应用技术领域。
背景技术
病毒诱导的基因沉默VIGS(Virus incluced gene silencing)是一种RNAi(RNAinterference)下调基因表达的方法。其主要原理是携带目标基因片段的病毒浸染宿主后,诱导宿主向下调控内源基因的表达水平并引起表型特征的变化,进而研究目标基因的功能。VIGS技术具有周期短、转化效率高、实验成本低等优点。该技术可在当代植株上呈现RNAi的效果,实现多个基因的同时沉默,尤其适用于转化体系不成熟和受基因型限制的物种。目前用于植物研究的VIGS载体主要有:烟草花叶病毒TMV(Tobacco mosaic virus)、烟草脆裂病毒TRV(Tobacco rattle virus)和马铃薯X病毒PVX(Potato virus X)等。
三七(Panaxnotoginseng(Burk.)F.H.Chen)为五加科(Araliaceae)人参属多年生草本植物,是云南省最具特色的中药材品种之一。三七在药用成分活性和育种实践等研究方面取得一定进展,但随着后基因组时代的到来,对大量基因功能鉴定需求日益迫切,关于生物学问题的最终阐明需要回归到特定物种上。目前,拟南芥、烟草和水稻等模式植物构建了稳定的遗传转化体系用于基因功能研究,但多年生植物由于其植物组织培养时间长、成本高和受基因型限制等原因,在构建稳定的遗传转化体系方面较为困难。三七作为多年生药用植物,虽然在基因组研究方面取得了一些进展,初步明确了调控药用活性成分合成和关键性状形成的目的基因,但由于其复杂的遗传背景和种群混杂等原因,在构建转化体系方面一直未取得突破,严重制约了对三七基因功能研究的进展。因此,构建一套不依赖遗传转化体系的基因沉默体系,将有望实现对三七相关功能基因的快速鉴定。
发明内容
本发明正是为了解决上述问题缺陷,提供一种病毒诱导的三七PDS基因沉默体系及应用。
本发明采用如下技术方案实现。
一种三七PnPDS基因,其核酸序列如SEQ ID NO.1所示。
一种沉默三七PnPDS基因的特异性核苷酸片段,其核苷酸序列如SEQ ID NO.2所示。
所述VIGS沉默载体为烟草脆裂病毒TRV2载体。
所述一种三七PnPDS基因VIGS沉默体系的构建方法按以下步骤进行:
1)设计沉默三七PnPDS基因的特异性核苷酸片段的特异性引物,并在上下游引物两端分别加上线性化pTRV2载体两个末端的同源序列(18-20bp)和酶切位点;
2)利用步骤1)中设计引物进行高保真PCR扩增目的基因;
3)将步骤2)中所获PCR产物与酶切后的pTRV2载体质粒进行同源重组,获得三七PnPDS基因的VIGS沉默体系的pTRV2-PnPDS;
所述沉默三七PnPDS基因的特异性核酸片段的扩增引物具体如下:
PnPDS-EcoRⅠ-F:
5′-TGAGTAAGGTTACCGAATTCACTCTGCATGCCTATTGC-3′
PnPDS-BamHⅠ-R:
5′-GTGAGCTCGGTACCGGATCCTGATCATATGTGTTCTTCAGTTTCCT-3′
所述的三七PnPDS基因的VIGS沉默体系在鉴定三七基因中的应用。
进一步的,所述的应用主要包括以下步骤:
(1)通过冻融法将pTRV2-PnPDS转化到农杆菌GV3101感受态细胞中,培养后进行PCR鉴定阳性;
(2)将分别含有pTRV1和pTRV2-PnPDS的OD=0.5、0.8、1.1的农杆菌菌液,分别按照体积比1:1混合,制备浸染液;
(3)采用真空渗透法对子叶期三七幼苗进行浸染;
(4)浸染后植株置于暗培养72h后转换为20℃/18℃,18h/6h光/暗周期下培养;
(5)观察基因沉默植株的表型变化、白化面积和通过荧光定量PCR检测被沉默目标基因的表达量。
本发明通过对子叶期的三七植株,进行不同浓度菌液的浸染,发现子叶期三七出现不同程度的白斑和白化现象,单株白化率高达44.52%。通过qPCR检测PnPDS的表达量,相对于对照,子叶期三七植株的PnPDS基因表达量皆显著降低。
本发明的有益效果为,(1)本发明首次实现了三七PnPDS基因VIGS体系的构建,获得了能使三七PnPDS基因沉默的体系;
(2)本发明构建的VIGS体系能够有效的降低三七PnPDS基因的表达,影响叶绿素的合成,使沉默叶片呈现漂白化表型,可用于三七白化植株材料的创制;
(3)本发明中三七VIGS沉默体系的建立为研究三七基因功能提供了有效的技术手段,同时增加了可使用VIGS技术的多年生植物种类。
下面结合附图和具体实施方式本发明做进一步解释。
附图说明
图1为三七PnPDS基因特异性片段扩增结果电泳图;(M:5000bp Maker)。
图2为pTRV2-PnPDS重组载体浸染三七真叶期叶片黄化后表型。A:注射法浸染真叶期三七叶片表型。白色箭头标记叶片注射位点。B:真空渗透浸染真叶期三七叶片表型。
图3为qRT-PCR检测真空渗透和注射浸染真叶期三七叶片PnPDS基因的表达水平。
图4为处理前三七子叶期幼苗形态。
图5为pTRV2-PnPDS重组载体真空浸染三七幼苗白化后表型和白化率。A:不同OD菌液处理后三七的白化叶片;B:不同OD菌液处理后三七单株白化叶片白化率统计。
图6为qRT-PCR检测真空浸染子叶期三七PnPDS基因的表达水平。
具体实施方式
结合具体实施案例对本发明进一步详细描述。下列实施例可为本技术领域普通技术人员进一步改进指南,并不以任何方式构成对本发明的限制。
在下述的实施例中,未注明的具体条件的实验方法,均为常规方法,按照本领域的公知手段实施。
如无特殊说明,以下实施例中的定量实验,均设置三次实验重复,结果取平均值。
实施例1
1.三七PnPDS基因VIGS沉默表达载体的构建
1.1PnPDS基因特异性片段的获得
结合三七基因组和转录组数据获得3条同源PnPDS的CDS序列信息,设计携带20bp同源片段和双酶切位点的引物。取三七幼嫩叶片,提取RNA,反转录得到cDNA。以cDNA为模板,使用携带线性化pTRV2载体两个末端的同源序列和酶切位点的引物对,对3条同源的PnPDS基因分别进行PCR扩增,结果如图1所示,只有PnPDS-2得到扩增产物。PnPDS-2扩增结果测序后如序列表的序列2所示。
PnPDS-1-EcoRⅠ-F:
5′-TCTGTGAGTAAGGTTACCGAATTCACCCGCAATTGAATCTCCTGC-3′
PnPDS-1-BamHⅠ-R:
5′-ACGCGTGAGCTCGGTACCGGATCCACAGTTCTCTTGACATTCATGGC-3′
PnPDS-2-EcoRⅠ-F:
5′-TGAGTAAGGTTACCGAATTCACTCTGCATGCCTATTGC-3′
PnPDS-2-BamHⅠ-R:
5′-GTGAGCTCGGTACCGGATCCTGATCATATGTGTTCTTCAGTTTCCT-3′
PnPDS-3-EcoRⅠ-F:
5′-TGAGTAAGGTTACCGAATTCATGGAGAAGGAAAGTGTCAACA-3′
PnPDS-3-BamHⅠ-R:
5′-GTGAGCTCGGTACCGGATCCTAGATTATGAACATTCGGGTAAGCC-3′
1.2重组病毒载体pTRV2-PnPDS的构建
使用Ready-to-Use Seamless Cloning Kit(生工)将上述获得的PCR产物与经过EcoRⅠ和BamHⅠ双酶切的pTRV2线性载体进行同源重组,热激法转化至大肠杆菌DH5α感受态细胞,挑取单克隆PCR菌液检测并测序验证,提取测序正确的重组质粒pTRV2-PnPDS。
将质粒pTRV1、pTRV2和pTRV2-PnPDS通过冻融法转化农杆菌GV3101感受态,挑选单克隆进行PCR菌液验证,选取PCR验证正确的阳性克隆进行摇菌,同时使用甘油保菌,于-80℃保存。
2.浸染植株
2.1材料准备
选取长势一致的一年生三七植株用于浸染实验。
2.2浸染液的制备
在LB抗性(50g/mL卡那霉素和50g/mL利福平)平板上挑选含有pTRV1、pTRV2和pTRV2-PnPDS质粒的阳性农杆菌单克隆,分别接种于2mL的LB液体抗性培养基中,28℃,200rpm摇床震荡培养约12h。然后以5%含量转接与LB抗性培养基中,28℃,200rpm摇床震荡培养至OD为0.8。6000rpm离心10min后收集各菌体,加入适量重悬液(10mM的MES,150M的乙酰丁香酮,10mM的MgCl2),于室温黑暗处静置3-5h后,将含有pTRV1与pTRV2空载(NEG)、pTRV1与pTRV2-PnPDS重组质粒的农杆菌分别按1:1体积混合,得到的浸染液后准备接种。
2.3浸染液真空渗透一年生三七植株叶片
将长势一致的三七植株分别倒置,浸泡在浸染液培养罐中,置于真空装置中,设置渗透压强为-50kPa,处理时间为10分钟,每组各抽真空1次,用无菌水冲洗3次。
2.4浸染液注射一年生三七植株叶片
将长势一致的一年生三七植株叶片使用灭菌水冲洗干净,后使用注射器吸取1mL渗透液注射三七叶片。
2.5浸染后植株培养
将抽真空和注射处理后植株置于黑暗条件下培养72h后转换为20℃/18℃,18h/6h光/暗周期下,光照为100-120mol/m2·s,相对湿度60~70%。
3.沉默植株表型观察
浸染处理25天后,对浸染植株(每组30株)进行表型观察。发现注射三七叶片大多坏死后干枯,未见黄化或白化表型(图2A);真空浸染pTRV2-PnPDS载体的三七植株叶片可见部分的黄化(图2B),但黄化面积较小。
观察统计各组中的沉默数量效率(沉默数量效率=有黄化表型植株/供试植株总数)。
表1各试验组沉默后情况
组别 | CK | NEG | pTRV2-PnPDS渗透组 | pTRV2-PnPDS注射组 |
沉默数量效率(%) | 0 | 0 | 3 | 0 |
4.qRT-PCR检测PnPDS基因表达水平
选取NEG、注射浸染pTRV2-PnPDS载体和真空浸染pTRV2-PnPDS载体的植株的黄化叶片,提取叶片总RNA,反转录为cDNA,以GAPDH为内参基因,在PnPDS沉默片段外区域设计引物对基因表达量进行qRT-PCR分析,基因表达量计算采用2-ΔΔct法。结果如图3所示,与空载相比,真空浸染pTRV2-PnPDS植株表达量有所降低,但未达到差异显著水平,结果表明PnPDS基因受到一定程度的沉默。但综合来看,其沉默效率低,黄化表型不具备典型性。
上述所涉及的荧光定量引物分别为:
qPnPDS-F:5′-GGTGGCTGCTTGGAAAGATG-3′
qPnPDS-R:5′-CATACGCCTGTCCACCGATA-3′
GAPDH-F:5′-TGGAATGGCCTTCCGAGTTC-3′
GAPDH-R:5′-CGTACCACGCGACAAGTTTC-3′
实施例2
1.三七PnPDS基因VIGS沉默表达载体的构建
1.1PnPDS基因特异性片段的获得
结合三七基因组和转录组数据获得PnPDS-2的CDS序列信息,设计携带20bp同源片段和双酶切位点的引物。取三七幼嫩叶片,提取RNA,反转录得到cDNA。以cDNA为模板,使用PnPDS-2-EcoRⅠ-F和PnPDS-2-BamHⅠ-R引物对进行PCR扩增,得到扩增产物。扩增结果测序后如序列表的序列2所示。
PnPDS-2-EcoRⅠ-F:
5′-TGAGTAAGGTTACCGAATTCACTCTGCATGCCTATTGC-3′
PnPDS-2-BamHⅠ-R:
5′-GTGAGCTCGGTACCGGATCCTGATCATATGTGTTCTTCAGTTTCCT-3′
1.2重组病毒载体pTRV2-PnPDS的构建
使用Ready-to-Use Seamless Cloning Kit(生工)将上述获得的PCR产物与经过EcoRⅠ和BamHⅠ双酶切的pTRV2线性载体进行同源重组,热激法转化至大肠杆菌DH5α感受态细胞,挑取单克隆PCR菌液检测并测序验证,提取测序正确的重组质粒pTRV2-PnPDS。
将质粒pTRV1、pTRV2和pTRV2-PnPDS通过冻融法转化农杆菌GV3101感受态,挑选单克隆进行PCR菌液验证,选取PCR验证正确的阳性克隆进行摇菌,同时使用甘油保菌,于-80℃保存。
2.浸染植株
2.1材料准备
选取三年生成熟饱满、大小均一的三七红籽,经人工洗去红色外果皮,使用5%CuSO4消毒处理30分钟后,蒸馏水清洗3次的三七种子作为实验材料。三七种子层积于含水量约25%的湿沙中,种子:湿沙约为1:3,置于20℃左右的黑暗条件下约45-55天,种子陆续萌发,选取长势一致的子叶期幼苗(图4)用于浸染实验。
2.2浸染液的制备
在LB抗性(50g/mL卡那霉素和50g/mL利福平)平板上挑选含有pTRV1、pTRV2和pTRV2-PnPDS质粒的阳性农杆菌单克隆,分别接种于2mL的LB液体抗性培养基中,28℃,200rpm摇床震荡培养约12h。然后以5%含量转接与LB抗性培养基中,28℃,200rpm摇床震荡培养至OD分别为0.5、0.8和1.1。6000rpm离心10min后收集各菌体,加入适量重悬液(10mM的MES,150M的乙酰丁香酮,10mM的MgCl2),于室温黑暗处静置3-5h后,将含有pTRV1与pTRV2空载(NEG)、pTRV1与pTRV2-PnPDS重组质粒的农杆菌分别按1:1体积混合,得到不同OD的浸染液后准备接种。
2.3真空渗透三七子叶期叶片
将长势一致的三七幼苗分别浸泡在OD=0.5、0.8和1.1和空载(NEG)的浸染液培养罐中,以水处理为空白对照(CK),置于真空装置中,设置渗透压强为-50kPa,处理时间为10分钟,每组各抽真空3次,用无菌水冲洗3次,后移栽至育苗盘中。
2.4浸染后植株培养
将抽真空后植株置于黑暗条件下培养72h后转换为20℃/18℃,18h/6h光/暗周期下,光照为100-120mol/m2·s,相对湿度60~70%。
3.沉默植株表型观察
浸染处理7天后,对植株成活率进行统计。处理20天后,浸染pTRV2-PnPDS载体的植株叶片开始出现白化表型(图5A),使用根系扫描仪对白化幼苗的白化率进行统计,发现OD=0.5处理组中单株白化率高达44.52%(图5B),且该表型可持续1-2月,而未侵染植株和NEG叶片没有任何变化,结果表明PnPDS基因沉默成功。
效果验证:
观察统计各组中在试验过程中的成活率(成活率=成活植株数/供试植株总数)和沉默数量效率(沉默数量效率=有白化表型植株/供试植株总数)。
表1各试验组培养情况
组别 | CK | NEG | PnPDS-0.5 | PnPDS-0.8 | PnPDS-1.1 |
成活率(%) | 78 | 72 | 76 | 68 | 67 |
沉默数量效率(%) | 0 | 0 | 21 | 15 | 9 |
4.qRT-PCR检测PnPDS基因表达水平
选取CK、NEG和不同OD浸染pTRV2-PnPDS载体的植株的白化叶片,提取叶片总RNA,反转录为cDNA,以GAPDH为内参基因,在PnPDS沉默片段外区域设计引物对基因表达量进行qRT-PCR分析,基因表达量计算采用2-ΔΔct法。结果如图6所示,与空载相比,pTRV2-PnPDS植株表达量均显著降低(P<0.05),表明本体系可以有效的用于鉴定三七基因功能。
上述所涉及的荧光定量引物分别为:
qPnPDS-F:5′-GGTGGCTGCTTGGAAAGATG-3′
qPnPDS-R:5′-CATACGCCTGTCCACCGATA-3′
GAPDH-F:5′-TGGAATGGCCTTCCGAGTTC-3′
GAPDH-R:5′-CGTACCACGCGACAAGTTTC-3′
以上内容表明三七中VIGS基因沉默体系构建成功。
传统基因敲除技术需要构建稳定的遗传转化体系,即使得到转基因植株也需要3年才能采收生产所用的三七种子,对于种质选育相关基因功能研究十分不利,本发明所述方法具有高速、方便操作和节约时间成本的特点。
与实施例1相比,本试验中三七沉默效率和白化率较显著提高,这可能是以一年生真叶期的三七叶片为试验材料时,三七叶片大小在生长过程基本没有变化,因此PnPDS基因的沉默对叶绿素合成影响较小。此外,由于三七叶片组织较薄,注射法浸染较真空浸染对三七机械伤害较大,植株无法正常生长后死亡,从而无法启动相应的基因沉默机制。
SEQ ID NO.1:
ATGTCTCAATTTGGCCAAGTCTCTGCAGTCAATTTGAGCACGCAAAGTAATGTAATAAACTTTTGGAACTCCCAATCTAATTGGGAATGTGGTGTTCATAGCGGTTCACGGCAGAGAAATGCACTATTCAGAGGTTGTTATTTTATGGGTCAAAGGGTGAAAATACCTATTGCGGATGCTCTGATAACAAGATCAAGAAAAAATGTAAACCGGTTGGAGGTGGTTTGCATTGACTATCCAAGACCAGAGATTGATAATACAGTTCCTTTCTTAGAAGCTGCTTACTTATCTTCATCCTTTCGTACTGCTCCCCGCCCAAATAAGCCACTGGAAATCGTAATTGCTGGTGCAGGGTTGGCTGGTTTATCTACTGCAAAATATTTGGCTGATGCAGGTCACAAGCCCATATTGTTGGAAGCAAGAGATGTTCTTGGTGGAAAGGTGGCTGCTTGGAAAGATGATGATGGAGACTGGTATGAGACTGGCTTACACATTTTCTTTGGGGCTTACCCGAATGTTCAGAACCTGTTTGGAGAACTAGGCATTAATGATCGATTGCAGTGGAAGGAGCATTCTATGATATTTGCGATGCCAAATAAGCCAGGGGAATTTAGCCGATTTGATTTTCCTGAAGTTCTACCTGCACCGTTAAATGGGATTTGGGCTATCTTGAAGAATAATGAAATGCTTACATGGCCTGAGAAAATCAAGTTTGCACTGGGACTCTTGCCAGCAATTATCGGTGGACAGGCGTATGTTGAGGCTCAAGATGGTTTAAGTGTCAAAGATTGGATGAGAAAGCAAGGTATACCAGATCGGGTTACTACTGAGGTTTTTGTTGCCATGTCGAAGTCATTAAACTTCATCAACCCAGATGAACTTTCAATGCAATGTGTTTTGATTGCTTTGAACCGATTTCTTCAGGAGAAGCATGGTTCAAAGATGGCTTTCTTAGATGGAAGCCCTCCAGAAAGACTCTGCATGCCTATTGCTGATCATATTCAGTCACTGGGTGGTGAAGTCCGGCTTAATTCACAAGTACAGAAGATCGAGCTAAATAACGATGGAACTGTGAAGAGTTTACTACTAACTAATGGGAATGTAATTGAAGCTGATGCATATGTAATTGCTGCTCCAGTTGATATCCTGAAGCTCCTTTTACCTGAAGACTGGAAGGAGATCCCATATTTCAGGAAATTGGATAAATTAGTTGGGGTCCCAGTTATCAATGTACATATATGGTTTGACAGGAAACTGAAGAACACATATGATCATCTACTTTTCAGCAGAAGCTCCCTTCTTAGTGTATATGCTGATATGTCTGTGACATGTAAGGAATATTATAACCCAAATCAATCCATGTTGGAGTTGGTTTTTGCACCTGCAGAAGAATGGATTTCACGAAGTGACACCGATATTATTGCTGCTACATTGAGTGAACTGGCAAGACTCTTTCCTGATGAGATTGGCCCGGATCAGAGTAAAGCAAAGATATTGAAGTATCATGTTGTTAAAACACCAAGATCTGTTTATAAAACTGTACCAGGCTGTGAACCCTGCCGTCCCTTGCAAAAATCTCCCATAGAGCGATTCTATCTAGCCGGCGATTACACAAAACAGAAGTATTTAGCTTCAATGGAGGGTGCTGTGCTCTCAGGAAAGCTTTGTGCACAAACTATTTTACAGGATTATGAGGTTCTTGTTTCCAGGGAGCAGAAGATGCTTGCTGAGGCAAGCGTTGTCTAA
SEQ ID NO.2:
ACTCTGCATGCCTATTGCTGATCATATTCAGTCACTGGGTGGTGAAGTCCGGCTTAATTCACAAGTACAGAAGATCGAGCTAAATAACGATGGAACTGTGAAGAGTTTACTACTAACTAATGGGAATGTAATTGAAGCTGATGCATATGTAATTGCTGCTCCAGTTGATATCCTGAAGCTCCTTTTACCTGAAGACTGGAAGGAGATCCCATATTTCAGGAAATTGGATAAATTAGTTGGGGTCCCAGTTATCAATGTACATATATGGTTTGACAGGAAACTGAAGAACACATATGATCA
以上所述的仅是本发明的部分具体实施例,方案中公知的具体内容或常识在此未作过多描述(包括但不仅限于简写、缩写、本领域惯用的单位)。应当指出,上述实施例不以任何方式限制本发明,对于本领域的技术人员来说,凡是采用等同替换或等效变换的方式获得的技术方案均落在本发明的保护范围内。本申请要求的保护范围应当以其权利要求的内容为准,说明书中的具体实施方式等记载可以用于解释权利要求的内容。
Claims (5)
1.一种三七PnPDS基因VIGS沉默体系的构建方法,其特征在于,所述构建方法包括如下步骤:1)设计沉默三七PnPDS基因的特异性核苷酸片段的特异性引物,并在上下游引物两端分别加上线性化pTRV2载体两个末端的同源序列和酶切位点;
2)利用步骤1)中设计引物进行高保真PCR扩增目的基因;
3)将步骤2)中所获PCR产物与酶切后的pTRV2载体质粒进行同源重组,获得三七PnPDS基因的VIGS沉默体系的pTRV2-PnPDS。
2.一种沉默三七PnPDS基因的特异性核苷酸片段,其特征在于,所述特异性核苷酸片段序列如SEQ ID NO.2所示。
3.检测权利要求2所述沉默三七PnPDS基因的特异性核苷酸片段的特异性引物,其特征在于,所述特异性引物如下:
PnPDS-EcoRⅠ-F:
5′-TGAGTAAGGTTACCGAATTCACTCTGCATGCCTATTGC-3′、
PnPDS-BamHⅠ-R:
5′-GTGAGCTCGGTACCGGATCCTGATCATATGTGTTCTTCAGTTTCCT-3′。
4.根据权利要求1所述方法的应用,其特征在于,所述应用为鉴定三七基因。
5.根据权利要求4所述应用的方法,其特征在于,该方法包括以下步骤:
(1)通过冻融法将pTRV2-PnPDS转化到农杆菌GV3101感受态细胞中,培养后进行PCR鉴定阳性;
(2)将含有pTRV1与pTRV2空载、pTRV1与pTRV2-PnPDS重组质粒的农杆菌菌液分别按1:1体积混合,制备浸染液;
(3)采用真空渗透法对子叶期三七幼苗进行浸染;
(4)浸染后植株置于暗培养72h后转换为20℃/18℃,18h/6h光/暗周期下培养;
(5)观察基因沉默植株的表型变化、白化面积和通过荧光定量PCR检测被沉默目标基因的表达量。
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