CN117025641B - 蒙古冰草八氢番茄红素脱氢酶基因AmPDS、特异性片段及应用 - Google Patents
蒙古冰草八氢番茄红素脱氢酶基因AmPDS、特异性片段及应用 Download PDFInfo
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Abstract
本发明涉及蒙古冰草八氢番茄红素脱氢酶基因AmPDS、特异性片段及应用,属于植物基因工程和基因编辑技术领域,蒙古冰草八氢番茄红素脱氢酶基因AmPDS的核苷酸序列如SEQ ID NO.1所示。本发明还提供蒙古冰草八氢番茄红素脱氢酶的特异性片段,其核苷酸序列如SEQ ID NO.2所示。本发明还提供一种重组病毒载体pTRV2‑AmPDS及其制备方法,利用所述重组病毒载体pTRV2‑AmPDS能够沉默蒙古冰草AmPDS基因,使蒙古冰草叶片变白。
Description
技术领域
本发明属于植物基因功能验证和植物基因工程技术领域,具体地涉及一种蒙古冰草八氢番茄红素脱氢酶基因AmPDS、特异性片段及应用。
背景技术
蒙古冰草(Agropyron mongolicum Keng),多年生禾本科牧草,茎秆直立疏松,有时基部横卧而节上生根成匍茎状,叶鞘短于节间,无毛,叶片常内卷或成扁平状,叶鞘比节短,紧密包茎;叶舌短,穗状花序,疏松,直线形;外稃呈卵状披针形,先端尖锐或呈芒状尖头,背部无毛或微毛,内稃与外稃等长,背有纤毛。蒙古冰草具有抗寒、抗旱、耐风沙、适口性好以及饲用价值高等优良特性。富含大量可为作物改良的抗性基因,在防风固沙,保持水土,改善生态环境中发挥着重要的作用。目前为止蒙古冰草在国内已在植物分类学、解剖学、牧草生物学、生理学、遗传育种学、栽培学以及人工草地建植、管理、利用等方面进行了研究。
采用传统的育种方法选育新品种,需要耗费大量的人力物力并且过程极为缓慢,随着分子生物学技术的发展,人们开始使用基因工程对植物进行改良,不断培育出符合人类期望和生产需求的新品种。近年来,随着分子生物学的发展,对植物进行功能基因研究成为热点,尤其是CRISPR/Cas9技术的产生,为分子育种奠定了基础,大大加快了作物分子育种的进程。但由于遗传转化体系的限制,植物基因功能的研究目前主要集中在一些模式植物上。
禾本科是第四大植物家族,包含有650到785种属,大约含有10000多种植物。所有的谷类作物和75%的栽培牧草都属于草类。虽然整个草家族很庞大,但是只有极少数的被开发作为牧草。蒙古冰草作为其中一员,目前,学者们已对蒙古冰草的细胞学、生理机制、转录组学、连锁图谱、QTL定位、遗传多样性等方面对蒙古冰草进行了研究,近几年蒙古冰草抗性基因挖掘也是研究的热点。然而,无论是通过各种育种手段改良新品种,还是通过体外技术快速繁殖筛选出新的变异植株,亦或是对功能基因的研究,都离不开高频、高效的组培遗传转化体系。植物遗传转化体系中,高效遗传转化受体系统的建立是转化过程中最关键的一步,因此建立一种有效的、稳定的再生系统和转化系统仍有许多问题需要进行细致深入的研究。农杆菌介导法是转基因技术中经常采用的一种手段,农杆菌介导的遗传转化技术在植物基因工程发展中是研究最清楚应用最广泛的,利用农杆菌在双子叶植株中的转化已经十分完善,其中的DNA能够转移到植物基因组上,从而使得植物得到转化。农杆菌介导的遗传转化过程是自然界发生的完美的基因转化过程,它的一段DNA能插入到受体植物细胞基因组,并稳定的遗传给后代,作为一种天然的植物基因转化系统,具有转化的外源DNA结构完整、转化机理清楚、整合位点稳定、拷贝数低、聚合后的外源基因结构变异较小等优点,因而备受重视。对于单子叶植物来说,对其遗传转化的研究开始较晚,主要在于单子叶植物不是农杆菌的天然宿主,早期农杆菌转化法比较适用于大多数双子叶植物和裸子植物,所以很多科学家认为农杆菌介导法不适合用于禾谷类植物和单子叶植物的遗传转化。但是随着农杆菌侵染机理研究的深入,农杆菌介导单子叶植物基因转化已经获得成功,近年来该方法在单子叶植物上取得了突破性进展,已成功获得了转基因玉米、水稻和小麦等禾本科作物。
基因沉默是真核生物细胞基因表达调控的一种重要手段,是植物自身防御病毒入侵的一种自然机制。病毒诱导的基因沉默(virus-induced gene silencing,VIGS)是利用病毒载体携带植物目的基因片段,通过各种方式侵染植物,重组病毒在植物中复制产生双链RNA(double strand RNA,dsRNA),在植物自身防卫反应转录后基因沉默(posttranscriptional gene silencing,PTGS)的机制作用下,dsRNA进一步产生siRNA(smallinterference RNA,siRNA),该产物能够诱导与其具有同源性的植物内源目的基因mRNA的降解,根据表型变异来进行基因功能分析,实现植物基因功能的快速鉴定。相比于传统的转基因技术,VIGS技术具有沉默效率高、周期短、操作简单、经济实惠等优点,并且不需要事先知道并克隆基因全长,也无需建立遗传转化体系,只需要一段300-500 bp的基因片段就能快速获得表型,进行基因功能的验证。目前,已经成为植物基因功能研究领域最具有吸引力的技术之一。
八氢番茄红素脱氢酶(PDS)是类胡萝卜素合成途径中的限制酶,参与植物类胡萝卜素的合成累积。PDS基因沉默会影响植物番茄红色的积累,番茄植株的绿色部分(叶子和绿色果实)因为缺乏该酶而导致叶绿体对光极度敏感,出现典型光漂白表型,因此变成白色。由于植物不同部位均出现容易观察的沉默表型,PDS在VIGS体系建立过程中得到广泛应用。
病毒诱导的基因沉默早期常用于烟草中,用来研究候选基因的功能,从而可以在不同的信号途径,尤其是抗逆途径中发现新基因。烟草脆裂病毒(tobacco rattlevirus,TRV)具有基因组较小,可以侵染植物生长点,不产生典型病毒性状,便于改造等优点,是目前在植物中应用最广泛、效果最好的VIGS病毒载体,对包括番茄、烟草、辣椒等在内的植物有良好的侵染效果,但尚未见到关于对蒙古冰草种子阶段的基因沉默的报道。
发明内容
本发明提供一种蒙古冰草AmPDS基因,通过构建含有AmPDS基因特异性片段的pTRV2病毒沉默表达载体,该载体经农杆菌介导转化蒙古冰草,诱导蒙古冰草内源AmPDS基因发生沉默,有效降低了蒙古冰草AmPDS基因的表达水平,导致白化表型。利用本发明所述方法能够高效快捷的直接转化蒙古冰草种子,并且能得到蒙古冰草的转化植株,该体系实现简单、快速,表型鉴定周期短、转化效率高,便于高通量操作,为大规模开展蒙古冰草基因功能研究提供有力工具。
本发明采用下述技术方案予以实现:
一种蒙古冰草八氢番茄红素脱氢酶基因AmPDS(Phytoene desaturase),其核苷酸序列如SEQ ID NO.1所示。
本发明还提供蒙古冰草八氢番茄红素脱氢酶的特异性片段,所述特异性片段的核苷酸序列如SEQ ID NO.2所示。
本发明还提供一种重组病毒载体pTRV2-AmPDS,所述重组病毒载体pTRV2-AmPDS含有所述的特异性片段。
本发明还提供所述重组病毒载体pTRV2-AmPDS的制备方法,所述方法为以蒙古冰草AmPDS为目的基因,是利用gateway重组克隆技术将AmPDS基因特异性片段连接至VIGS病毒骨架载体pTRV2的MCS区域的BamHⅠ中,将连接产物转化DH5a感受态,利用JC pTRV2-F和JCpTRV2-R引物进行菌液PCR鉴定,并送擎科公司测序,比对测序结果,获得构建重组病毒载体pTRV2-AmPDS。
本发明还提供利用重组病毒载体pTRV2-AmPDS沉默蒙古冰草AmPDS基因的应用,所述的应用方法为通过冻融法将pTRV1、pTRV2、重组病毒载体pTRV2-AmPDS转化到农杆菌EHA105感受态细胞,选择全剥种皮与半剥种皮蒙古冰草种子消毒处理,无菌萌发的种子与农杆菌EHA105侵染液混合后,经过40min抽真空、8min超声、55min抽真空侵染处理,所用真空抽滤的压力为0.1MPa;将种子与侵染液在28℃共培养14h后,倒去上层菌液,将侵染后的种子捞出放滤纸上吹干,然后将侵染后的种子在无糖固体培养基上共培养1周后,转移至固体培养基上继续培养;
将蒙古冰草种子实验材料置于温度27℃,光照强度2000lx,光/暗周期14 h/10h的条件下生长。
进一步,所用真空抽滤的压力为0.1MPa;
进一步,所述的无糖固体培养基为MS基础培养基中含有50mg·L-1赤霉素、400mg·L-1特美汀和4g·L-1 Phytagel,pH=5.95;
进一步,所述的固体培养基为MS基础培养基中含有50mg·L-1赤霉素,400mg·L-1特美汀,4g·L-1蔗糖,4g·L-1 Phytagel,PH=5.95。
本发明与现有技术相比的有益效果:
1)本发明首次获得的蒙古冰草AmPDS基因,并发现了能够沉默蒙古冰草AmPDS基因表达的特异性片段,构建了蒙古冰草AmPDS基因VIGS沉默体系。
2)本发明通过烟草脆裂病毒构建蒙古冰草AmPDS基因沉默体系,能够在蒙古冰草植株整体进行系统扩散传播。
3)本发明所述沉默体系能够有效降低蒙古冰草AmPDS基因的表达水平,类胡萝卜素合成途径被阻断,从而使得植物产生光漂白现象,即叶绿素被漂白,叶片变白。
4)本发明由农根菌介导,通过根癌农杆菌TRV1与TRV2混合侵染系统导入外源基因并获得转基因植株,达到全株基因表达量水平降低;首先在植物种子培养过程中,消毒后的无菌萌发,可以确保种子萌发不受其他胁迫或竞争的影响;其次根癌农杆菌侵染种子会对种子的生长发育造成一定的负影响,本发明在每种培养基中都加入适量的赤霉素,来促进种子萌发,缩短萌发周期,提高转化效率。本发明中无糖固体培养基培养相对其他方式(土培、水培)培养能够更好的保证在植物正常生长发育的前提下,植物生长不受其他菌类的影响或影响较小;本发明中有糖固体培养基能够保证种子在生长发育阶段为种子提供足够的碳源和能量。
此外,在建立蒙古冰草基因沉默体系的前提下,还可利用此体系对蒙古冰草中一些未知功能基因,也可对重组病毒载体pTRV2-AmPDS的AmPDS所在的MCS区域位置进行替换,在蒙古冰草中进行功能验证,以期为加速挖掘蒙古冰草优异基因、改良蒙古冰草种质提供加速工具。
附图说明:
图1蒙古冰草AmPDS基因组DNA序列与CDS序列的琼脂凝胶电泳图。
A.蒙古冰草AmPDS基因组DNA序列琼脂凝胶电泳图;
B.蒙古冰草AmPDS的CDS序列琼脂凝胶电泳图
图2构建重组病毒载体pTRV2-AmPDS农杆菌菌液PCR的琼脂糖凝胶电泳图。M:DNAladder DL2000;1-X:农杆菌克隆
A.构建重组病毒载体pTRV2-AmPDS的SnapGene图谱
B.构建重组病毒载体pTRV2-AmPDS农杆菌菌液PCR的琼脂糖凝胶电泳图。M:DNAladder DL5000;L1-L7:农杆菌克隆。
图3三种不同处理的种子萌发率的统计图;
图4三种不同处理的种子污染率的统计图;
图5对转化后蒙古冰草转化效率与阳性率的统计图;
图6实时荧光定量PCR(qRT-PCR)检测蒙古冰草AmPDS的沉默效果图。
A.qRT-PCR分析转化后的蒙古冰草单株中AmPDS表达量水平情况(CK:
AmPDS:重组病毒载体pTRV2-AmPDS侵染的蒙古冰草幼苗。)图中WT为
CK对照组材料,B1、B2为pTRV1与pTRV2-AmPDSh混合侵染材料
B.PCR分析蒙古冰草阳性植株中重组载体pTRV2-AmPDS的存在情况;
图中1:DNAmarker DL5000;L1:从根癌农杆菌(EH105)菌落扩增的PCR产物;L2:从非转化植株基因组DNA扩增的PCR产物;L3:去离子水扩增的PCR产物;L4-L7从蒙古冰草再生植株基因组DNA扩增的PCR产物。
具体实施方式:
下面结合附图和实例对本发明做进一步详细说明。
实施例1:AmPDS全场基因序列获得;
在NCBI网站(https://www.ncbi.nlm.nih.gov/)获得番茄Tomato PDS(GenBank
#88683)基因核苷酸序列、小麦PDS(FJ517553.1)等基因核苷酸序列,由于蒙古冰草没有全基因组序列,所以我们以它的近缘种的PDS序列进行BLAST检索,发现PDS序列具有较高的保守度,由此我们通过同源克隆的方式,利用PDS基因的保守序列设计引物在蒙古冰草中进行克隆,并获得全长基因序列,将其命名为AmPDS,核苷酸序列如SEQ ID NO.1所示。
实施例2:AmPDS基因特异性片段的克隆
1)用于沉默蒙古冰草八氢番茄红素脱氢酶基因(PDS)的AmcPDS基因特异性片段。取蒙古冰草整株幼苗,在1.5ml离心管加入小钢珠,用液氮冷冻并研磨成粉状,然后用Aidlab试剂盒操作步骤提取RNA,-80℃冻存备用。取出-80℃冻存的RNA,按照TEANS反转录试剂盒两步法操作步骤合成cDNA第一链,于-20℃保存。
2)反转录PCR反应体系为:RNA模板6μL、Random Primer 1μL、AnchoredOligo(dT)18Primer 1μL,65℃5min;②R-MIX 10μL、E-MIX 1μL、Remover 1μL42℃1h,85℃15s;cDNA模板2μL、上下特异性游引物各1μL、Phanta 10μL,补ddH2O至20μL扩增条件为:95℃变性3min、95℃15s、68℃30s 35个循环、72℃延伸7min。
3)八氢番茄红素脱氢酶基因AmPDS的全长基因序列为模板,设计用于扩增AmPDS基因CDS结构域上的369bp特异性片段的引物AmPDS-F和AmPDS-R;上述引物序列分别为:
AmPDS-F:5’-CTGACGAGTTATCCATGCAGTGCA-3’
AmPDS-R:3’-ATATATGAACATTGATGACAGGAAC-5’
扩增体系:
扩增反应程序:
PCR扩增AmPDS基因特异性片段,其大小为369bp,核苷酸序列如SEQ IDNO.4所示。
实施例3:重组病毒载体pTRV2-AmPDS的构建;
1)以pTRV2质粒为模板,利用BamHⅠ限制性内切酶对pTRV2进行酶切,使用诺威赞胶回收试剂盒回收,所得线性化载体记作pTRV2-BamH I切。
上述酶切反应体系为:
BamH I酶切反应反应程序:
37℃60min
2)以蒙古冰草AmPDS为目的基因,是利用gateway重组克隆技术将AmPDS基因特异性片段连接至VIGS病毒骨架载体pTRV2的MCS区域的BamHⅠ中,将连接产物转化DH5a感受态,利用JC pTRV2-F和JC pTRV2-R引物进行菌液PCR鉴定,并送擎科公司测序,比对测序结果,获得构建重组病毒载体pTRV2-AmPDS,如图2。
上述引物序列分别为:
JC pTRV2-F:5’-ATGTTCAGGCGGTTCTTGTG-3’
JC pTRV2-R:3’-TTAAGAAACTTTATTGCCAA-5’
检测反应体系:
检测反应程序:
实施例4:通过冻融法将pTRV1、pTRV2、重组病毒载体pTRV2-AmPDS转化到农杆菌EHA105感受态细胞中:
通过冻融法将pTRV1、pTRV2、重组病毒载体pTRV2-AmPDS转化到农杆菌EHA105感受态细胞中:根癌农杆菌EH105感受态细胞,冰上融化后,加入5 uL的pTRV1、pTRV2、重组病毒载体pTRV2-AmPDS质粒,冰上静置30分钟,液氮中冷冻1分钟;然后37℃水浴3 min加入950μL无抗生素的LB液体培养基,28℃,200 rpm,振荡培养3小时;离心浓缩菌液,用100μL LB液体培养基回溶菌体,后将回溶后的菌体涂于加固体LB培养基(Kan,50μg·mL-1;Rif,50μg·mL-1),28℃下培养,长出单克隆后,保存阳性根癌农杆菌落,对载体pTRV1、载体pTRV2、重组病毒载体pTRV2-AmPDS进行PCR鉴定;PCR反应:青岛擎科生物技术有限公司合成的JCpTRV2-F:5’-ATGTTCAGGCGGTTCTTGTG-3’,JC pTRV2-R:3’-TTAAGAAACTTTATTGCCAA-5’,进行PCR扩增,PCR反应总体系为20μL,加入JC pTRV2-F、JC pTRV2-R各1μL,模板DNA2μL,2×MIXBuffer10μL,6μL的去离子水补齐20μL反应总体系。PCR反应条件:95℃预变性5分钟;95℃变性15秒,50℃退火30秒,72℃延伸1分30秒,29个循环;72℃延伸5分钟,然后取出PCR反应产物4℃恒温保存。取得PCR扩增产物,进行琼脂糖凝胶电泳(1.0%)并拍照,点样时5μL5000 bp LadderMaker作为分子量标准。如图2。将构建重组病毒载体pTRV2-AmPDS大肠杆菌接菌至新的LB液体培养基中(Kan,50μg·mL-1),28℃,200 rpm,振荡培养过夜,利用诺威赞质粒提取试剂盒提取构建重组病毒载体pTRV2-AmPDS质粒。
实施例5:蒙古冰草种子消毒处理及无菌苗的获得
1)种子种皮处理:蒙古冰草种子在生长时,胚根与胚芽最先在种子底部胚的位置破皮而出,所以为了提高种子的发芽率和更好的提高种子自身与消毒后的无菌率,以蒙古冰草的种子为材料,对种子种皮进行了三种不同的处理方式①打磨处理:用镊子将种子固定,使用砂纸对种子胚位置进行打磨,直至磨去种皮露出被包被的种子;②半剥处理:在胚芽与胚根破皮生长的种胚所在位置,将其种皮剥去露出被包被的种子③全剥处理:将整个种子的种皮剥去,只剩种子。
2)无菌苗的获得:在超净工作台中,将1)中三种不同种皮处理的蒙古冰草种子在分别放置于三个放入无菌培养皿种,其依次进行75%酒精消毒30S、无菌水冲洗三次、20%NaClO消毒20min、无菌水冲洗三次、0.5%H2O2消毒0.5%、无菌水冲洗的处理,消毒后在铺设三层浸湿的滤纸上进行液体无菌培养,萌发至第4天,整体种子萌发完毕,作为侵染材料,种子无菌萌发的环境条件:温度为27℃,光周期为24h黑暗,观察并统计种子的萌发率如图3;污染率如图4所示。对三不同处理种皮后消毒的萌发率与污染率进行了统计,将两因素结合进行分析,得到的结果显示萌发率统计中半剥种皮处理与打磨种皮处理的种子萌发率较高,但打磨种皮处理污染率最高,所以选择全剥种皮与半剥种皮为后面侵染材料。
实施例6:蒙古冰草幼苗病毒侵染
1)重组病毒载体转化农杆菌:以pTRV1质粒、pTRV2质粒和pTRV2-AmPDS重组病毒载体分别转化农杆菌EH105后,挑取新鲜培养转化子单菌落,分别接种到1mLLB液体培养基(Kan,50μg·mL-1;Rif,50μg·mL-1)中,28℃180转min-1培养12 h;然后转入50mLLB液体培养基(Kan,50μg·mL-1;Rif,50μg·mL-1)中,28℃180转min-1培养12-16 h;培养至终浓度为OD600=0.6;将携带有pTRV1载体的农杆菌EH105菌液分别与pTRV2载体的农杆菌EH105菌液、携带pTRV2-AmPDS重组病毒载体的农杆菌EH105菌液的培养液按体积比1:1混匀制成2种混合菌液,并补充乙酰丁香酮(Acetosyringone)(19.62mg·L-1)、半胱氨酸(Cysteine)(400mg·L-1)、吐温-20(TWEEN-20)(5ml·L-1)用于侵染蒙古冰草种子。
2)根癌农杆菌介导的TRV病毒基因沉默侵染:无菌萌发的种子与侵染液混合后,在经过40min抽真空、8min超声、55min抽真空侵染处理后,所用真空抽滤的压力为0.1MPa;将种子与侵染液在28℃共培养14h后,倒去上层菌液,将侵染后的种子捞出放滤纸上吹干以除去过多的附着根癌农杆菌,期间需不断翻种子至干燥;然后将侵染后的种子在无糖固体培养基上共培养1周后,转移至固体培养基上继续培养;
将蒙古冰草种子实验材料置于温度27℃,光/暗周期14 h/10 h的条件下生长。
所述的无糖固体培养基:MS基础培养基,50mg·L-1赤霉素,400 mg·L-1特美汀,4g·L-1 Phytagel,PH=5.95;
所述的固体培养基:MS基础培养基,50mg·L-1赤霉素,400 mg·L-1特美汀,4g·L-1蔗糖,4g·L-1Phytagel,PH=5.95。
3)种子固体培养基及培养条件:将步骤7)的种子置于培养条件为22℃光照强度2000lx,光周期为14小时光照,10小时黑暗条件下生长,期间统计种子污染情况如图4、观察新长出的蒙古冰草叶片的表型变化,并根据表型判断提取植株RNA,检测植株单株AmPDS的表达情况、统计蒙古冰草污染率(图5)与转化效率(图6),即构建得到蒙古冰草AmPDS基因序列沉默体系;
实施例7:蒙古冰草白化苗PCR检测
1)蒙古冰草白化苗DNA提取:取新鲜的蒙古冰草白化苗植株叶片剪碎置于1.5 mL离心管中,加入2×CTAB提取液1 mL,使用小研杵捣碎后充分混匀,65℃水浴60分钟,在水浴期间轻轻颠倒离心管几次。水浴结束后,待液体冷却至室温加入等体积的氯仿剧烈震荡15秒,12000 rpm室温离心10分钟,取上清液转移到新的1.5 mL离心管中,然后加入等体积的异丙醇震荡混匀,于-20℃冰箱中沉淀30分钟,12000 rpm室温离心10分钟。然后用1 mL70%乙醇洗涤沉淀,7500rpm离心5分钟,倒掉上清洗涤液。洗涤操作重复1次,最后自然干燥DNA,去除残余乙醇溶液。加入去离子水溶解DNA,超微量仪测定DNA浓度和比值。
2)PCR反应:青岛擎科生物技术有限公司合成的JC pTRV2-F:5’-ATGTTCAGGCGGTTCTTGTG-3’,JC pTRV2-R:3’-TTAAGAAACTTTATTGCCAA-5’,进行PCR扩增,PCR反应总体系为20μL,加入JC pTRV2-F、JC pTRV2-R各1μL,模板DNA2μL,2×MIX Buffer10μL,6μL的去离子水补齐20μL反应总体系。PCR反应条件:95℃预变性5分钟;95℃变性15秒,50℃退火30秒,72℃延伸1分30秒,29个循环;72℃延伸5分钟,然后取出PCR反应产物4℃恒温保存。取的PCR扩增产物,进行琼脂糖凝胶电泳(1.0%)并拍照,点样时5μL 5000 bpLadderMaker作为分子量标准。通过白化苗DNA的胶图观察得到检测其体内pTRV2-AmPDS重组病毒载体的PCR产物大小分为1083bp(图6 L4-L7)符合预期。
实施例8:蒙古冰草AmPDS基因的功能鉴定
侵染后蒙古冰草AmPDS基因表达量测定:固体培养3周,观察新长出的蒙古冰草叶片的表型变化,叶片表型为白色,取其叶片与根,用RNA试剂盒提取叶片总RNA,以Actin为内参基因,通过RT-PCR检测被沉默后目标基因的表达量。所用引物为:AmPDS-RT-F:AGTTCGACCTCCCTTGGCTT;
AmPDS-RT-R:ATCCTGCACCAGCAATCACG;
Actin-RT-F:CAATGGGAAGCAAGGCTGTAA;
Actin-RT-R:AACAATCCGAACTGAGGCAATC。
蒙古冰草中AmPDS基因表达量测定结果见图5,图5结果表明:与对照CK相比,受到携带pTRV2-AmPDS重组病毒载体农杆菌侵染的蒙古冰草中AmPDS基因表达量显著降低。
总之本发明成功地建立一种以蒙古冰草AmPDS基因VIGS沉默的基因快速筛选体系,该体系实现简单、快速、高通量分析鉴定蒙古冰草AmPDS基因功能,为VIGS技术体系在蒙古冰草上的大规模应用奠定了基础。也为其他单子叶植物使用TRV-VIG的抽真空-超声-抽真空的侵染方式提供了应用依据。利用本发明所述方法能够高效快捷的直接转化蒙古冰草种子,并且能得到蒙古冰草的转化植株,有望大大缩短蒙古冰草基因遗传转化时基因自身基因筛选时长,加速蒙古冰草遗传转化、植物改造进程,为大规模开展蒙古冰草基因功能研究提供有力工具。蒙古冰草八氢番茄红素脱氢酶基因AmPDS(Phytoene desaturase):ATGGATACCAGCTGCCTATCATCTATGAACATAGCTGGAGCGAAGCAAGTAAGATCTTTTGCTGGACAACTTCATACACAGAGGTGCTTCACAAGTAGCAGTGTCCAAGCACTAAAAACTAGTCACCGTACGACCTTTAGTTCGACCTCCCTTGGCTTTAGGAATAAAGTAAAAGGATCACGCCGTGGACTTCGTGCTCTGCAGGTTGTTTGCCAAGATTTTCCAAGGCCTCCACTAGAGAACACGATTAACTATTTGGAAGCTGGCCAGCTTTCTTCGTCGTTTAGAAGCAGTGAACGCCCCAGTAAACCATTACAGGTCGTGATTGCTGGTGCAGGATTGGCTGGTCTATCAACTGCAAAATACCTGGCAGATGCTGGCCATAAACCCATAGTGCTTGAGGCAAGAGATGTGTTGGGCGGAAAGTTGGCTGCTTGGAAGGATGAAGATGGTGATTGGTATGAGACTGGCCTTCATATTTTTTTTGGAGCTTATCCCAATGTACAGAATTTGTTTGCTGAGCTTGGTATTAGTGATCGCTTGCAATGGAAGGAACACTCCATGATATTTGCCATGCCAAACAAACCAGGAGAATACAGCCGTTTTGATTTCCCAGAGACTTTGCCGGCGCCCTTAAATGGAGTGTGGGCCATACTGAAAAACAATGAAATGCTTACTTGGCCGGAAAAGGTGAAGTTTGCTATTGGGCTTCTACCGGCAATGCTTGGTGGCCAAGCTTACGTTGAAGCTCAAGATGGCTTAACTGTTTCAGAATGGATGGAAAAGCAGGGTGTTCCTGATCGAGTCAACGATGAGGTTTTTATTGCAATGTCCAAGGCGCTCAATTTCATAAACCCTGACGAGTTATCCATGCAGTGCATTCTGATTGCTCTAAACCGATTTCTCCAGGTACAACTTCCGTTCCTCTATTCCTCCTGGAGACATAGTTGACATAAATGTGTAGAAGATGCAAACATTCGTTCACACAATCACACCATAACGACAACTTGGGGGTATTACTTAATGAAAAAACTGTGTAAATGTGTAGGAGACACATGGCTCGAAAATGGCATTCTTGGATGGCAATCCTCCTGAAAGGCTATGCATGCCTATTGTTAACCACATTCAGTCTTTGGGTGGTGAGGTCCGGCTGAATTCTCGTATTCAGAAAATTGAACTGAACCCTGACGGAACTGTGAAGCACTTTGCACTTACTGATGGGACTCAAATAACTGGAGATGCATATGTTTGTGCAGCACCAGGTGCGATTTATTTTCAAGAATCATGCTTTGCACCTATTCAGTTTAACTGACTAGCTTGTGATTCAGTCGATATCTTCAAGCTTCTTGTACCACAAGAGTGGAGAGAGATCTCTTATTTCAAAAGGCTGGATAAGTTGGTGGGAGTTCCTGTCATCAATGTTCATATATGGTTAGTTGATTGA。特异性片段SEQ ID NO.2:
CTGACGAGTTATCCATGCAGTGCATTCTGATTGCTCTAAACCGATTTCTCCA
GGTACAACTTCCGTTCCTCTATTCCTCCTGGAGACATAGTTGACATAAATGT
GTAGAAGATGCAAACATTCGTTCACACAATCACACCATAACGACAACTTGG
GGGTATTACTTAATGAAAAAACTGTGTAAATGTGTAGGAGACACATGGCTC
GAAAATGGCATTCTTGGATGGCAATCCTCCTGAAAGGCTATGCATGCCTATT
GTTAACCACATTCAGTCTTTGGGTGGTGAGGTCCGGCTGAATTCTCGTATTC
AGAAAATTGAACTGAACCCTGACGGAACTGTGAAGCACTTTGCACTTACTG
ATGGGACTCAAATAACTGGAGATGCATATGTTTGTGCAGCACCAGGTGCGAT
TTATTTTCAAGAATCATGCTTTGCACCTATTCAGTTTAACTGACTAGCTTGTG
ATTCAGTCGATATCTTCAAGCTTCTTGTACCACAAGAGTGGAGAGAGATCTC
TTATTTCAAAAGGCTGGATAAGTTGGTGGGAGTTCCTGTCATCAATGTTCATATATGGTTAGTTGATTGA。
Claims (7)
1.一种蒙古冰草八氢番茄红素脱氢酶基因AmPDS,其特征在于,其核苷酸序列如SEQ IDNO.1所示。
2.一种核酸分子,其特征在于,所述核酸分子为蒙古冰草八氢番茄红素脱氢酶的特异性片段,所述特异性片段的核苷酸序列如SEQ ID NO.2所示。
3.一种重组病毒载体pTRV2-AmPDS,其特征在于,所述重组病毒载体pTRV2-AmPDS含有权利要求2所述的核酸分子。
4.权利要求3所述重组病毒载体pTRV2-AmPDS的制备方法,其特征在于,所述方法为以蒙古冰草AmPDS为目的基因,利用gateway重组克隆技术将权利要求2所述的核酸分子连接至VIGS病毒骨架载体pTRV2的MCS区域的BamHⅠ中,将连接产物转化DH5a感受态,进行菌液PCR鉴定,比对测序结果,获得构建重组病毒载体pTRV2-AmPDS,AmPDS基因的核苷酸序列如SEQ ID NO.1所示。
5.利用权利要求3所述重组病毒载体pTRV2-AmPDS在沉默蒙古冰草AmPDS基因中的应用,其特征在于,所述应用为通过冻融法将pTRV1、pTRV2、重组病毒载体pTRV2-AmPDS转化到农杆菌EHA105感受态细胞,选择全剥种皮或半剥种皮的蒙古冰草种子消毒处理,无菌萌发的种子与农杆菌EHA105侵染液混合后,经过40min抽真空、8min超声、55min抽真空侵染处理,所用抽真空的压力为0.1MPa;将种子与侵染液在28℃共培养14h后,倒去上层菌液,将侵染后的种子捞出放滤纸上吹干,然后将侵染后的种子在无糖固体培养基上培养1周后,转移至固体培养基上继续培养;置于温度27℃,光照强度2000lx,光/暗周期14h/10h的条件下生长;
所述的半剥种皮为在胚芽与胚根破皮生长的种胚所在位置,将其种皮剥去露出被包被的种子。
6.根据权利要求5所述的应用,其特征在于,所述的无糖固体培养基为MS基础培养基中含有50mg·L-1赤霉素、400mg·L-1特美汀和4g·L-1Phytagel,pH=5.95。
7.根据权利要求5所述的应用,其特征在于,所述的固体培养基为MS基础培养基中含有50mg·L-1赤霉素,400mg·L-1特美汀,4g·L-1蔗糖,4g·L-1Phytagel,pH=5.95。
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