CN109867715A - 一种叶绿体蛋白和ATPase酶活性突变体在提高植物抗逆性中的应用 - Google Patents
一种叶绿体蛋白和ATPase酶活性突变体在提高植物抗逆性中的应用 Download PDFInfo
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Abstract
本发明提供了一种苔藓PpHsp70基因和PpHsp70基因编码的叶绿体蛋白及其ATPase酶突变体PpHsp70m在提高植物耐高温、耐旱、耐盐胁迫等综合抗逆性中的应用。本发明通过对PpHsp70基因及其酶活突变体PpHsp70m在苔藓和水稻中的过表达转基因植株进行分子鉴定和胁迫试验,检测PpHsp70和PpHsp70m基因表达量变化及抗性改变等,结果发现与野生型相比,PpHsp70和PpHsp70m基因过表达转基因植株分别对高温、干旱和盐胁迫更耐受。这说明PpHsp70基因编码的蛋白质及其酶活突变体是影响植物综合抗逆性的关键因子,能够应用于植物抗逆品质改良中。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种PpHsp70基因和PpHsp70基因编码的叶绿体蛋白及其ATPase酶突变体PpHsp70m在提高植物综合抗逆性中的应用。
背景技术
环境压力造成植物细胞水平上大量蛋白质机能失调,细胞广泛采取的最有效修复措施之一是热激蛋白(又叫伴侣蛋白,Hsps)与变性蛋白结合,维持蛋白质的可溶状态,帮助解折叠蛋白质重新组装成有活性的构像(2000)。最初是在热激条件下发现这类特异性蛋白的积累,直至今天,人们认识到它们所参与的细胞活动远远不止在热激处理下维护蛋白质的稳定构像。在植物中的研究显示,超量表达Hsp26显著增强拟南芥和水稻的耐热性(Xue et al.,2010;Kim et al.,2012)。玉米叶绿体sHsp26在高温和干旱联合处理下显著提高,干涉实验的蛋白质组分析表明sHsp26能够稳定叶绿体蛋白质组和PSII的蛋白质构像增强玉米的耐旱性(Hu et al.,2010)。小立碗藓中至今仅一例关于Hsps参与非生物逆境胁迫的报道来自于PpHsp16.4,该基因响应脱落酸(ABA)、盐胁迫和水杨酸诱导,蛋白质通常以低聚复合体的形式聚集在叶绿体周围,对其研究证明小立碗藓的抗逆能力得自于逆境中保护细胞完整性逆境后修复重建机制(Ruibal et al.,2013)。
Hsp70是真核生物广泛存在的一类伴侣蛋白,进化上结构和功能高度保守,主要行使帮助其他蛋白质折叠和组装、阻止蛋白质错误聚合、和帮助蛋白质跨膜转运的功能(Stetler et al.,2010)。Hsp70可以参与蛋白质折叠的内稳态以及与ATP结合和水解紧密结合的蛋白相互作用影响整个蛋白系统的稳定性(Preissler et al.,2017),还会参与MEP途径中第一步酶错误折叠聚合后的降解,维持细胞功能的正常发挥(Llamas et al.,2017)。Hsp70家族在果蝇的发现已超过半个世纪(Ritossa,1962),近年来人们逐渐认识到Hsp70家族在抗逆境方面的作用,玉米幼苗经100μM ABA处理后H2O2水平迅速升高导致细胞质Hsp70显著累积引起抗旱性和抗高温能力增强(Hu et al.,2010)。动物细胞(Guo etal.,2007)或植物细胞(Dat et al.,2000;Mittler,2002)内均发现H2O2的积累会导致Hsp70家族成员大量表达,显示Hsp70参与细胞防护工作。Hsp70作为水解酶,具有水解ATP底物的能力,与Hsp40、GrpE组成一个系统共同完成前体蛋白质转运工作,Hsp40负责招募前体蛋白形成复合体,结合到Hsp70/ATP完成水解反应,所产生的能量驱动底物进入叶绿体基质GrpE协助释放出ADP,新的ATP进入Hsp70,开始下一个循环(Liu et al.,2014)。改变Hsp70水解酶的活性,是改变蛋白质活性和功能的一个重要方法。
苔藓是最古老的陆生植物之一,是水生到陆生的过度植物,分化于4亿5千万年前,在进化地位上位于藻类之后、蕨类和种子植物之前,和微管植物属于单起源系统上的姐妹进化支,具有极其重要的研究地位。全世界约有苔藓植物21200种,遍布除海洋外的地球上每个角落,甚至生长于荒漠、冻原及岩石上,耐旱、耐寒、耐贫瘠,适应力极强,是大自然的先锋与拓荒者(Kidron,2014;曹同等,2014)。以苔藓进行逆境适应性研究已经成为抗逆研究的热点之一(Doherty et al.,2017),而利用苔藓来源的基因进行其他高等植物的抗逆改良及研究也在进行中(Liu et al.,2017)。以小立碗藓为对象开展的基因组学和分子生物学研究发现,为适应陆地生活,小立碗藓获得耐干旱基因、耐高温基因、感光基因、紫外修复基因等陆地胁迫响应基因(Rabara et al.,2013;Rensing et al.,2008)。对我国多省大型铜矿和金矿生态环境破坏严重地区的植物资源调查发现,苔藓作为先锋植物在这些维管植物无法存活的地方生长。因此利用苔藓植物作为基因供体,具有普遍适应性,同时苔藓植物与其他高等植物共生的互不影响性,也为其在作物改良中的应用奠定了基础。
可惜的是目前真正应用苔藓伴侣蛋白基因进行抗逆性遗传改良的案例非常少,能改良综合抗逆性的例子更是未见报道。本发明首次利用叶绿体伴侣蛋白和其酶活性改变突变体获得了多种植物的综合抗性材料,将为植物抵抗非生物胁迫,应对环境多样性变化和遗传工程改良提供理论和实践基础。
发明内容
本发明的目的在于对小立碗藓的叶绿体热激蛋白基因进行开发利用,提供一种PpHsp70基因或ATPase酶活性改变突变体在提高植物抗逆性中的应用。
为了实现本发明的上述目的,本发明提供了如下的技术方案:
PpHsp70基因或PpHsp70基因编码的叶绿体蛋白及其ATPase酶活性突变体在提高植物抗逆性中的应用。
PpHsp70基因或PpHsp70基因编码的叶绿体蛋白及其ATPase酶活性突变体在提高植物耐高温、耐旱、耐盐胁迫中的应用。
根据所述的应用,其中所述PpHsp70基因的核苷酸序列如SEQ ID No.1所示。
根据所述的应用,其中所述PpHsp70基因编码的蛋白质的氨基酸序列如SEQ IDNo.2所示。
根据所述的应用,其中所述PpHsp70基因的ATPase酶突变体在原始PpHsp70第271个氨基酸位点发生了苏氨酸到丙氨酸T271A突变。
根据任一项所述的应用,其中所述提高植物耐高温、耐旱、耐盐胁迫性是通过下述方法步骤得以实现的:
(1)将前面所述编码序列连接于植物表达调控序列,形成植物表达载体;
(2)将所述植物表达载体通过PEG介导的原生质体法导入小立碗藓,通过农杆菌侵染的方法转入水稻植物细胞,筛选得到转化细胞;
(3)将所述转化细胞进行植株再生,鉴定后得到转基因阳性植株;
(4)将所述转基因阳性植株进行抗逆性筛选和评价。
根据所述的应用,其中所述植物为小立碗藓和水稻。
根据所述的应用,其中所述抗逆性为抗高温、抗干旱和抗高盐。
本发明同时还提供了一种提高植物抗逆性的方法,该方法包括如下步骤:
(1)将PpHsp70基因ATP结合区第271个碱基点突变后获得PpHsp70m,测试ATPase酶活性;
(2)将PpHsp70和PpHsp70m连接于植物表达调控序列,形成植物重组表达载体,分别为小立碗藓表达载体和水稻表达载体;
(3)将所述植物重组表达载体通过PEG介导的原生质体转化方法导入小立碗藓细胞,筛选得到转化植株;
(4)将所述植物重组表达载体通过农杆菌介导的方法转入水稻植物愈伤组织,筛选得到转化植株;
(5)将所述得到的PpHsp70和PpHsp70m基因过表达的小立碗藓植株进行抗逆性筛选,获得抗性指标;
(6)将所述得到的PpHsp70和PpHsp70m基因过表达的水稻植株T0代收获得到的种子发芽后苗期进行抗逆性筛选,获得抗性指标。
与现有技术相比,本发明的有益效果在于:
本发明提供了一种PpHsp70和ATPase酶活突变体PpHsp70m基因或基因编码的蛋白质在提高植物抗逆性中的应用。本发明通过对PpHsp70过表达转基因植株进行分子鉴定和非生物逆境胁迫试验,检测其基因表达量及抗性改变等。结果发现:与野生型相比,小立碗藓过表达转基因植株对高温、干旱、高盐处理后的恢复能力更强,水稻过表达植株对逆境的耐受力有不同程度提高。说明PpHsp70和PpHsp70m基因或基因编码的蛋白质可用于多种植物抗逆性的改良。
附图说明
图1为本发明实施例1所述PpHsp70和PpHsp70m蛋白ATPase酶活性。PpHsp70和PpHsp70m蛋白质的ATPase酶活性测定结果显示PpHsp70m(TA)活性增强20%。横坐标为反应溶液中使用的ATP底物浓度。数据来源于三次生物学重复。“*”表示t测验的P值小于0.05,差异显著;“**”表示t测验的P值小于0.01,差异极显著。数据的差异性分析是与PpHsp70在同一种底物浓度下酶活性做对比。
图2为本发明实施例1所述PpHsp70和PpHsp70m基因过表达小立碗藓植株在DNA和RNA水平下的鉴定结果。过表达小立碗藓植株的鉴定结果。A,转基因植株DNA水平PCR检测结果,左起第一泳道为DNA Marker,第二泳道为阳性质粒对照。第三泳道为阴性对照。其他泳道为4个独立转化单株。B,转基因植株RNA水平qPCR检测结果。数据来源于三次生物学重复。
图3为本发明实施例1所述PpHsp70和PpHsp70m基因过表达小立碗藓植株逆境(高温,干旱和0.5mM高盐)处理响应下叶绿素荧光参数Fv/Fm的变化,对比野生型对照,过表达植株的光合参数比较高,说明在各种逆境下都有不同程度的恢复能力提高。PpHsp70和PpHsp70m基因过表达小立碗藓植株逆境(高温,干旱和0.5mM高盐)处理响应下叶绿素荧光参数Fv/Fm的变化,对比野生型对照,过表达植株的光合参数比较高,说明在各种逆境下都有不同程度的恢复能力提高。数据来源于三次生物学重复。“*”表示t测验的P值小于0.05,差异显著;“**”表示t测验的P值小于0.01,差异极显著。数据的差异性分析是与对照同一种处理条件下的参数做对比。
图4为本发明实施例1所述PpHsp70和PpHsp70m基因过表达水稻植株在DNA和RNA水平下的鉴定结果。过表达水稻植株的鉴定结果。A,转基因植株DNA水平PCR检测结果,左起第一泳道为DNA Marker,第二泳道为阴性对照。第三泳道为阳性质粒对照。其他泳道为3个独立转化单株。B,转基因植株RNA水平qPCR检测结果。数据来源于三次生物学重复。
图5为本发明实施例1所述PpHsp70和PpHsp70m基因过表达水稻植株逆境处理响应下植株表型变化,对比野生型对照,过表达植株对不同逆境的耐受力有所提高。A,高温、干旱处理下植株存活率统计;B,0.2mM高盐处理下株高统计。PpHsp70和PpHsp70m基因过表达水稻植株逆境处理响应下植株表型变化,对比野生型对照,过表达植株对不同逆境的耐受力有所提高。A,高温、干旱处理下植株存活率统计;B,0.2mM高盐处理下株高统计。数据来源于三次生物学重复。“*”表示t测验的P值小于0.05,差异显著;“**”表示t测验的P值小于0.01,差异极显著。数据的差异性分析是与对照同一种处理条件下的参数做对比。
图6为本发明所述植物表达调控pPOG1和pCAMBIA2300载体图。
具体实施方式
本发明提供了一种PpHsp70和第271个突变体PpHsp70m基因或基因编码的蛋白质在提高植物抗逆性中的应用。在本发明中,所述PpHsp70基因为生物中普遍存在的热激蛋白类编码基因,来源于小立碗藓叶绿体。所述PpHsp70基因的核苷酸序列优选如SEQ ID No.1所示。所述PpHsp70基因编码的蛋白质的氨基酸序列优选如SEQ ID No.2所示。所述SEQ IDNo.2所示的表达蛋白包括一段N-端的400个氨基酸的三磷酸腺苷(ATPase)功能域。本发明通过对PpHsp70和ATPase酶突变体PpHsp70m过表达转基因植株进行分子鉴定和胁迫试验,检测其基因表达量情况及抗性改变等。发现与野生型相比,过表达转基因植株对高温、干旱、盐胁迫更耐受。说明PpHsp70和PpHsp70m基因或基因编码的蛋白质可用于植物抗逆性品种的改良。
本发明所述PpHsp70和PpHsp70m基因以及基因编码的蛋白质在提高植物抗逆性中的应用优选包括以下步骤:
(1)设计构建包含的PpHsp70和PpHsp70m基因的载体pCAMBIA2300和pPOG1。
(2)酶切步骤:通过酶切将(1)中所述的pCAMBIA2300和pPOG1酶切获得pCAMBIA2300*和pPOG1*基因片段;
(3)将PpHsp70和PpHsp70m基因片段与所述步骤(2)中获得的载体pCAMBIA2300*和pPOG1*基因片段连接,获得表达载体pCAMBIA2300-PpHsp70,pCAMBIA2300-PpHsp70m,pPOG1-PpHsp70和pPOG1-PpHsp70m;
(4)利用PEG介导的原生质体转化的方法,将表达载体pPOG1-PpHsp70和pPOG1-PpHsp70m转化到小立碗藓的细胞中,经过筛选获得过表达PpHsp70和PpHsp70m的再生植株;利用农杆菌介导的方法,分别将表达载体pCAMBIA2300-PpHsp70和pCAMBIA2300-PpHsp70m通过农杆菌浸泡的方法侵染水稻愈伤组织,经过清洗与筛选获得抗性愈伤组织,而后得到过表达PpHsp70和PpHsp70m的再生植株。
(5)得到抗性植株后进行DNA水平及RNA水平的鉴定,获得阳性植株。
(6)获得阳性植株后的抗逆性鉴定方法,将获得的过表达的再生植株进行高温、干旱、高盐耐受能力的测试。
下面结合实施例对本发明提供的PpHsp70和PpHsp70m基因以及基因编码的蛋白质在提高植物抗逆性中的应用进行详细的说明,但是不能把它们理解为本发明保护范围的限定。
实施例1
本发明所使用目的基因PpHsp70为来源于小立碗藓的热激蛋白编码基因,其编码序列如SEQ ID No.1所示,PpHsp70基因编码的蛋白质的氨基酸序列如SEQ ID No.2所示。PpHsp70m来源于用PpHsp70在第271个氨基酸位点苏氨酸(Thr)到丙氨酸(Ala)的定点突变。
实施例2
ATPase酶T271A突变体PpHsp70m的产生。
从小立碗藓RNA反转录获得的cDNA片段上扩增得到PpHsp70的cDNA序列,然后通过TA试剂盒(大连宝生物生物工程有限公司)连接到T载体,成为T-Hsp70。扩增引物:PpHsp70m-F:ATGGCATCCGCGGTGGGATC(SEQ ID No.3)PpHsp70m-R:CGTTGAATCAGTGAAGTCTG(SEQ ID No.4)。通过QuikChange kit(美国Stratagene)试剂盒利用PCR的方法,产生T-Hsp70在第271个氨基酸位点苏氨酸(Thr)到丙氨酸(Ala)的定点突变,最终获得PpHsp70m。PCR扩增引物序列如下:QCTAF:CTTGGCGGGGGCGCCTTTGATGTTTC;QCTAR:GAAACATCAAAGGCGCCCCCGCCAAG。
实施例3
ATPase活性测定及结果。
将小立碗藓PpHsp70和PpHsp70m基因利用SpeI和BamHI内切酶构建到pTYB12载体上,将该表达载体转化到BL21(DE3)感受态细胞(北京全式金生物TransGen Biotech)中。Hsp70蛋白经过1mM的异丙基β-D-1-硫代半乳糖苷诱导后使用New England Biolabs的几丁质粉按照试验说明进行纯化。纯化蛋白使用Amicon Ultra-4离心过滤装置浓缩纯化蛋白,然后蛋白质浓度通过Bradford assay,利用BSA作为标样进行测试。ATPase的活性通过改造后的孔雀石绿法进行测定(Chang et al.,2008)。该方法具体步骤如下:制备一个包含梯度ATP浓度(0to 10mM),DnaK,Hsp70(0.3mM),DnaJ(1mM),GrpE(1mM),prSSU(100nM),以及NR合成底物(NRLLLTG,200nM;上海捷瑞生物工程有限公司合成)的反应体系到一个总体积25μl的反应缓冲液中。这个反应缓冲液包含40mMHEPES-KOH(pH 7.0),75mM KCl,以及4.5mM Mg(CH3COO)2。在37℃孵育1h后,加入80μl孔雀绿试验液(孔雀绿(0.081%[w/v]):聚乙烯乙醇(2.3%[w/v]):仲钼酸铵四水化合物(5.7%[w/v]使用6M HCl):水=2:1:1:2)至反应体系当中。而后加入10μl 34%的柠檬酸钠终止非酶促水解作用。把所有反应体系混合后在37℃孵育15min后进行吸光度测试,选择波长620nm。测定结果如图1所示,PpHsp70m活性比原始蛋白增强20%。
实施例4
表达载体的构建。
(1)克隆基因PpHsp70的编码核苷酸序列片段:
小立碗藓载体构建引物:PpHsp70-PpF:gcggccgc ATGGCATCCGCGGTGGGATCTGTGG,PpHsp70-PpR:gtcgac TCACGTTGAATCAGTGAAGTCTGCA。
水稻载体构建引物:PpHsp70-F:ggtaccATGGCATCCGCGGTGGGATCPpHsp70-R:tctagaCGTTGAATCAGTGAAGTCTG。
模板:小立碗藓基因组DNA,采用CTAB法提取。
扩增体系和程序:使用南京诺唯赞公司的高保真聚合酶:Phata Master(p505),体系(50μl):F(10μM)1μl,R(10μM)1μl,Phata Master1μl,2*buffer 25μl,dNTP 1μl,H2O 21μl。使用Eppendorf Master PCR仪,程序:95℃预变性5min,95℃变性20s,58℃退火30s,72℃延伸2.5min(扩增效率1kb/min),35个循环,循环后72℃延伸5min,4℃保温。扩增结束后进行电泳回收目的片段。
(2)将克隆获得的PpHsp70基因的DNA片段通过酶切链接的方法,连接到通过37℃KpnI和XbaI及NotI和SalI内切酶双酶切获得的载体pCAMBIA2300*和pPOG1*片段上,连接使用Thermo公司的T4连接酶,4℃过夜的条件下进行连接,后期转化大肠杆菌及筛选阳性克隆使用通用的方法。
实施例5
小立碗藓稳定转化。
采用PEG介导的原生质体转化的方法转化小立碗藓,将生长5天左右的小立碗藓原丝体材料经过2%的崩溃酶裂解成原生质体,然后在PEG介导下于45℃进行热激,得到的转化细胞经过间隔一次复苏培养的两次抗性筛选进行筛选,得到待选择的转化子材料。整个转基因过程从转化到获得转化子材料一般需要2-4个月的时间。
实施例5
水稻稳定转化。
采用农杆菌介导的侵染水稻愈伤组织的方法进行转化,本研究采用农杆菌EHA介导的高效转化体系,将成熟的种子去壳消毒后在诱导培养基上26℃暗培养30-40天;诱导出的愈伤在继代培养基上扩大培养3次,每次继代培养大约为15天;挑选鲜黄而硬实的愈伤在26℃预培养3-5天后用农杆菌EHA105菌液浸泡30分钟进行浸染,19℃共培养2天,无菌水清洗愈伤数遍后转至加筛选标记抗生素的筛选培养基上,本发明中使用载体的抗性为新霉素(NptII),使用抗性浓度为20mg/L的MS培养基,经过2-3次筛选,每次15天左右,获得抗性愈伤;抗性愈伤转到预分化培养基上培养7天,然后在光照条件下分化培养基上分化出再生植株;再生植株在生根培养基上光照培养7天诱导生根;最后经3天炼苗后移栽。整个转基因过程从种子到转基因苗移栽一般需要4-5个月的时间。该转基因方法为已发表的成熟方法(Hiei et al.,Plant J,1994,6:271-282)。结果如图4所示,在阳性植株中基因的表达量显著高于野生型对照。
实施例6
DNA及RNA水平筛选方法。
采用CTAB法对获得的再生植株进行总DNA的提取,DNA水平鉴定引物为
PpHsp70-DF:ATGGCATCCGCGGTGGGATC,
PpHsp70-DR:ACATCAAAGGTGCCCCCGCC。
采用Trizol法对获得的再生植株进行总RNA进行提取,并使用北京全式金生物工程有限公司的反转录试剂盒One-Step gDNA Removal and cDNA SynthesisSuperMix进行cDNA合成,之后进行半定量PCR测定。RNA水平鉴定引物为PpHsp70-RF:GTTGTTGCGTACACGAAGAA,PpHsp70-RR:AGAAACTTGCTTGGACTCAT。
PCR扩增体系和程序:使用Transgen公司的TransStart Tip Green qPCRSuperMix进行扩增,优选体系为(50μl)cDNA模板1μl,F(10μM)0.4μl,R(10μM)0.4μl,2×TransStart Tip Green qPCR SuperMix 10μl,H2O加至20μl。使用Eppendorf Master PCR仪,程序:95℃预变性30s,95℃变性5s,58℃退火30s,72℃延伸30s,40个循环,循环后72℃延伸5min,4℃保温。结果如图2和4所示,在阳性植株中基因的表达量显著高于野生型对照。
实施例7
阳性植株对多种逆境胁迫的表现。
获得小立碗藓阳性植株后,对获得的阳性材料进行培养,培养至生长40天的配子体阶段,进行抗性测试,高温胁迫处理是将生长材料的培养皿放入45℃培养箱,处理6小时后恢复生长2天时观察植物生长状态,测试叶绿素荧光Fv/Fm参数;干旱处理是将生长至40天的配子体材料直接挑取暴露于空气中进行空气干燥,处理1小时后恢复生长2天观察植物生长状态,测试叶绿素荧光Fv/Fm参数;盐胁迫是将生长40天的配子体材料移入含有0.5mMNaCl的水培液体中,处理5小时后恢复生长2天观察植物生长状态,测试叶绿素荧光Fv/Fm参数。用IMAGING-PAM FluorImager和Imaging Win软件测量植物的叶绿素荧光参数。在测量Fv/Fm之前,植物应暗适应最少30min。
结果如图3所示,对比野生型对照,过表达植株在不同逆境处理后恢复能力有所提高。
获得水稻阳性植株后,进行抗性能力测试的方法具体步骤如下:在获得再生植株后收获其T0代种子,种子催芽后,在正常条件下生长至四叶期时进行各种逆境处理,高温胁迫是将幼苗生长盆放入42℃后,处理1天后恢复培养7天观察植物生长状态并检测存活率;干旱处理是将幼苗生长盆干旱处理7天,复水7天观察植物生长状态并检测存活率;盐胁迫是添加0.2mM NaCl溶液到幼苗生长盆,7天后观察植物生长状态并检测株高。结果如图5所示,在高温和干旱处理下,对照植株仅有约21%恢复,而过表达植株约有42%得到了恢复,显著高于对照(t测验,P<0.05)。高盐处理下,过表达植株长势明显强于对照植株,株高显著高于对照45%(t测验,P<0.05)。
由此可见,本发明提供的提高植物抗逆能力的方法,是通过获得稳定表达PpHsp70基因并编码PpHsp70蛋白质的植株,通过PpHsp70的作用,从而提高植物抗逆性能力。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国科学院昆明植物研究所
<120> 一种叶绿体蛋白和ATPase酶活性突变体在提高植物抗逆性中的应用
<170>中国国家知识产权局
<160> 2
<210> 1
<211> 2124
<212> DNA
<213> 小立碗藓(Physcomitrella patens)
<400> 1
atggcatccg cggtgggatc tgtggcattc ttgccctgtt cagttgcagg acaggggagt 60
gtcgtctaca aagttcgctt ttcaaagcat cggaaatgca cattgcgtgg tcagaagcct 120
tcggatagag ccttcttcgg aggggatttt tctatttcaa gaagtactca ggaatgcaat 180
tcaaagcatg aaaggagctc aaggcctctt cgtgtaactg ccgaaaaagt tgtcggcatt 240
gatcttggaa caacaaactc tgcaatggca gctatggaag gtggacagcc tacaataatt 300
actaattctg agggccagag gacaacccct tcggttgttg cgtacacgaa gaaaggtgat 360
cgattagtgg ggcagattgc taagcgccaa gctgtagtga accctgcgaa cacattttac 420
tcagtcaagc gattcattgg acggaaaatg aacgaagtgg aagatgagtc caagcaagtt 480
tcttaccaag tcattcgaga ctccaatggt aatgtgaaac tcgattgtcc agcaatcaac 540
aagcagtttg ctgctgaaga aatatccgcg caggttctaa gaaaattggt agacgatgcc 600
agcaagtttc taagcgacaa agttgacaag gctgttataa cagtgcctgc ttacttcaac 660
gacagtcaac gccaggctac taaagatgct ggtcgaattg ctggaattga tgtactgaga 720
atcatcaacg aacccactgc ggcatcgttg gcatatggtt tcgacaagaa gaaaaacgaa 780
actatcttgg tctttgacct tggcgggggc acctttgatg tttcagtctt ggaagttgga 840
gatggggttt ttgaagtact ttccacagcg ggagacaccc atctcggtgg tgacgacttt 900
gacaagagaa tagtggattg gctggcgaaa gaattcaaag ccgctgaagg cattgacctc 960
ttaaaggatg ctcaagcact tcagaggctg actgaaagtg ctgaaaaagc taagattgaa 1020
ttatcctccc tcactcagac aactatcagt cttcctttta ttacagcgac tgcggaggga 1080
cctaagcaca ttgatacaac acttacacgt gccaagtttg aggagttgtg ttcggatctc 1140
ttagacagat gtcgtgagcc agtaaagaga gctttagacg atgccaagtt gagtctcaag 1200
gatttgcagg aagttgtctt ggtgggaggg tcgactcgaa ttcctagtgt gcagcaattg 1260
gtcaagcaga tgacaggaaa agatccaaat gttactgtta atcctgatga agttgttgct 1320
cttggtgcag cagttcaggc tggagttcta tctggtgagg ttggtaatat tgtgcttctg 1380
gatgtttcgc ctctatcgct aggactggaa actttggggg gtgtcatgac aaagatcatt 1440
cctcgaaata cgacactgcc gacttccaag aaagaggtct tctctactgc ggctgatggt 1500
caaacaagcg tcgagattaa tgtgctccag ggagaacgtg agtttgtaag ggataataaa 1560
tcgctcggaa gcttcaggtt ggatgggatt gtacctgcac cacgtggagt tccacaaatc 1620
gaggtgacct ttgatattga tgccaatggc attttatcgg tcacagctat ggacaaagga 1680
tcaggaaaga agcaggacat ccagataact ggtgcctcca ccctcaacaa agatgatgtg 1740
gagagaatgg tccaagaagc agaaagaaat gcagaagaag ataagaaacg cagggaggtt 1800
attgatttaa aaaatcagag tgatagtatg atttatcagg cagagaagca gttgaaggaa 1860
ctgagtgaca aagttccagc agacctgaag agtcgtgtgg aaacgaaggt ggtcgatctc 1920
aaggaagccg cgaagacaga ggatgtggaa aaaatgaaaa gagctcaaga agcgttacaa 1980
caagaagtaa tgcaaattgg acaagcaata tatggaagcg gcgctgcaca cgcaggccct 2040
gcacaacctg gttccggagc tgcttcatca cctcctggag atgatgctga ggtgattgat 2100
gcagacttca ctgattcaac gtga 2124
<210> 2
<211> 707
<212> PRT
<213> 小立碗藓(Physcomitrella patens)
<400> 2
Met Ala Ser Ala Val Gly Ser Val Ala Phe Leu Pro Cys Ser Val
1 5 10 15
Ala Gly Gln Gly Ser Val Val Tyr Lys Val Arg Phe Ser Lys His
20 25 30
Arg Lys Cys Thr Leu Arg Gly Gln Lys Pro Ser Asp Arg Ala Phe
35 40 45
Phe Gly Gly Asp Phe Ser Ile Ser Arg Ser Thr Gln Glu Cys Asn
50 55 60
Ser Lys His Glu Arg Ser Ser Arg Pro Leu Arg Val Thr Ala Glu
65 70 75
Lys Val Val Gly Ile Asp Leu Gly Thr Thr Asn Ser Ala Met Ala
80 85 90
Ala Met Glu Gly Gly Gln Pro Thr Ile Ile Thr Asn Ser Glu Gly
95 100 105
Gln Arg Thr Thr Pro Ser Val Val Ala Tyr Thr Lys Lys Gly Asp
110 115 120
Arg Leu Val Gly Gln Ile Ala Lys Arg Gln Ala Val Val Asn Pro
125 130 135
Ala Asn Thr Phe Tyr Ser Val Lys Arg Phe Ile Gly Arg Lys Met
140 145 150
Asn Glu Val Glu Asp Glu Ser Lys Gln Val Ser Tyr Gln Val Ile
155 160 165
Arg Asp Ser Asn Gly Asn Val Lys Leu Asp Cys Pro Ala Ile Asn
170 175 180
Lys Gln Phe Ala Ala Glu Glu Ile Ser Ala Gln Val Leu Arg Lys
185 190 195
Leu Val Asp Asp Ala Ser Lys Phe Leu Ser Asp Lys Val Asp Lys
200 205 210
Ala Val Ile Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln
215 220 225
Ala Thr Lys Asp Ala Gly Arg Ile Ala Gly Ile Asp Val Leu Arg
230 235 240
Ile Ile Asn Glu Pro Thr Ala Ala Ser Leu Ala Tyr Gly Phe Asp
245 250 255
Lys Lys Lys Asn Glu Thr Ile Leu Val Phe Asp Leu Gly Gly Gly
260 265 270
Thr Phe Asp Val Ser Val Leu Glu Val Gly Asp Gly Val Phe Glu
275 280 285
Val Leu Ser Thr Ala Gly Asp Thr His Leu Gly Gly Asp Asp Phe
290 295 300
Asp Lys Arg Ile Val Asp Trp Leu Ala Lys Glu Phe Lys Ala Ala
305 310 315
Glu Gly Ile Asp Leu Leu Lys Asp Ala Gln Ala Leu Gln Arg Leu
320 325 330
Thr Glu Ser Ala Glu Lys Ala Lys Ile Glu Leu Ser Ser Leu Thr
335 340 345
Gln Thr Thr Ile Ser Leu Pro Phe Ile Thr Ala Thr Ala Glu Gly
350 355 360
Pro Lys His Ile Asp Thr Thr Leu Thr Arg Ala Lys Phe Glu Glu
365 370 375
Leu Cys Ser Asp Leu Leu Asp Arg Cys Arg Glu Pro Val Lys Arg
380 385 390
Ala Leu Asp Asp Ala Lys Leu Ser Leu Lys Asp Leu Gln Glu Val
395 400 405
Val Leu Val Gly Gly Ser Thr Arg Ile Pro Ser Val Gln Gln Leu
410 415 420
Val Lys Gln Met Thr Gly Lys Asp Pro Asn Val Thr Val Asn Pro
425 430 435
Asp Glu Val Val Ala Leu Gly Ala Ala Val Gln Ala Gly Val Leu
440 445 450
Ser Gly Glu Val Gly Asn Ile Val Leu Leu Asp Val Ser Pro Leu
455 460 465
Ser Leu Gly Leu Glu Thr Leu Gly Gly Val Met Thr Lys Ile Ile
470 475 480
Pro Arg Asn Thr Thr Leu Pro Thr Ser Lys Lys Glu Val Phe Ser
485 490 495
Thr Ala Ala Asp Gly Gln Thr Ser Val Glu Ile Asn Val Leu Gln
500 505 510
Gly Glu Arg Glu Phe Val Arg Asp Asn Lys Ser Leu Gly Ser Phe
515 520 525
Arg Leu Asp Gly Ile Val Pro Ala Pro Arg Gly Val Pro Gln Ile
530 535 540
Glu Val Thr Phe Asp Ile Asp Ala Asn Gly Ile Leu Ser Val Thr
545 550 555
Ala Met Asp Lys Gly Ser Gly Lys Lys Gln Asp Ile Gln Ile Thr
560 565 570
Gly Ala Ser Thr Leu Asn Lys Asp Asp Val Glu Arg Met Val Gln
575 580 585
Glu Ala Glu Arg Asn Ala Glu Glu Asp Lys Lys Arg Arg Glu Val
590 595 600
Ile Asp Leu Lys Asn Gln Ser Asp Ser Met Ile Tyr Gln Ala Glu
605 610 615
Lys Gln Leu Lys Glu Leu Ser Asp Lys Val Pro Ala Asp Leu Lys
620 625 630
Ser Arg Val Glu Thr Lys Val Val Asp Leu Lys Glu Ala Ala Lys
635 640 645
Thr Glu Asp Val Glu Lys Met Lys Arg Ala Gln Glu Ala Leu Gln
650 655 660
Gln Glu Val Met Gln Ile Gly Gln Ala Ile Tyr Gly Ser Gly Ala
665 670 675
Ala His Ala Gly Pro Ala Gln Pro Gly Ser Gly Ala Ala Ser Ser
680 685 690
Pro Pro Gly Asp Asp Ala Glu Val Ile Asp Ala Asp Phe Thr Asp
695 700 705
Ser Thr
707
Claims (9)
1.PpHsp70基因或PpHsp70基因编码的叶绿体蛋白及其ATPase酶活性突变体在提高植物抗逆性中的应用。
2.PpHsp70基因或PpHsp70基因编码的叶绿体蛋白及其ATPase酶活性突变体在提高植物耐高温、耐旱、耐盐胁迫中的应用。
3.根据权利要求1或2所述的应用,其特征在于,所述PpHsp70基因的核苷酸序列如SEQID No.1所示。
4.根据权利要求1或2所述的应用,其特征在于,所述PpHsp70基因编码的蛋白质的氨基酸序列如SEQ ID No.2所示。
5.根据权利要求1或2所述的应用,其特征在于,所述PpHsp70基因的ATPase酶突变体在原始PpHsp70第271个氨基酸位点发生了苏氨酸到丙氨酸T271A突变。
6.根据权利要求1~5任一项所述的应用,其特征在于,所述提高植物耐高温、耐旱、耐盐胁迫性是通过下述方法步骤得以实现的:
(1)将权利要求3和5所述编码序列连接于植物表达调控序列,形成植物表达载体;
(2)将所述植物表达载体通过PEG介导的原生质体法导入小立碗藓,通过农杆菌侵染的方法转入水稻植物细胞,筛选得到转化细胞;
(3)将所述转化细胞进行植株再生,鉴定后得到转基因阳性植株;
(4)将所述转基因阳性植株进行抗逆性筛选和评价。
7.根据权利要求6所述的应用,其特征在于,所述植物为小立碗藓和水稻。
8.根据权利要求6所述的应用,其特征在于,所述抗逆性为抗高温、抗干旱和抗高盐。
9.一种提高植物抗逆性的方法,其特征在于该方法包括如下步骤:
(1)将PpHsp70基因ATP结合区第271个碱基点突变后获得PpHsp70m,测试ATPase酶活性;
(2)将PpHsp70和PpHsp70m连接于植物表达调控序列,形成植物重组表达载体,分别为小立碗藓表达载体和水稻表达载体;
(3)将所述植物重组表达载体通过PEG介导的原生质体转化方法导入小立碗藓细胞,筛选得到转化植株;
(4)将所述植物重组表达载体通过农杆菌介导的方法转入水稻植物愈伤组织,筛选得到转化植株;
(5)将所述得到的PpHsp70和PpHsp70m基因过表达的小立碗藓植株进行抗逆性筛选,获得抗性指标;
(6)将所述得到的PpHsp70和PpHsp70m基因过表达的水稻植株T0代收获得到的种子发芽后苗期进行抗逆性筛选,获得抗性指标。
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