CN114774378A - 调控植物花粉管与柱头识别的硫氧还蛋白及其制备方法 - Google Patents
调控植物花粉管与柱头识别的硫氧还蛋白及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种调控植物花粉管与柱头识别的硫氧还蛋白及其制备方法,所述硫氧还蛋白的序列表如SEQ ID No.1或SEQ ID No.2所示。所述硫氧还蛋白为含有序列表SEQ ID No.1或SEQ ID No.2的真核重组表达质粒pETrx转化大肠杆菌DH5α获得的重组蛋白。通过用上述序列表的硫氧还蛋白能够有效提升花粉在柱头表面的水合和萌发以及花粉管在柱头表面的生长。此外,本申请还提供了一种调控植物花粉管与柱头识别的硫氧还蛋白的制备方法,该方法能够精确的制得硫氧还蛋白,方便硫氧还蛋白的使用。
Description
技术领域
本发明涉及生物技术领域,尤其是涉及一种调控植物花粉管与柱头识别的硫氧还蛋白及其制备方法。
背景技术
植物授粉过程包括花粉在柱头上的黏附、水合、萌发出花粉管、花粉管在花柱中极性生长、花粉管到达珠孔释放精细胞与卵细胞、中央细胞融合等,这个过程受到许多基因精细而复杂的调控。授粉过程中,花粉(管)与柱头细胞的识别决定花粉在柱头表面的水合和萌发以及花粉管在柱头表面的生长。
因此,如何通过花粉(管)与柱头细胞的识别,以提升花粉在柱头表面的水合和萌发以及花粉管在柱头表面的生长,成为亟待解决的问题。
发明内容
本发明的目的在于提供一种调控植物花粉管与柱头识别的硫氧还蛋白及其制备方法,以提升花粉在柱头表面的水合和萌发以及花粉管在柱头表面的生长。
为了实现上述目的,本发明实施例提供如下技术方案:
根据本发明实施例的第一方面,提供一种调控植物花粉管与柱头识别的硫氧还蛋白,所述硫氧还蛋白的序列表如SEQ ID No.1或SEQ ID No.2所示。
进一步的,所述硫氧还蛋白为含有序列表SEQ ID No.1或SEQ ID No.2的真核重组表达质粒pETrx转化大肠杆菌DH5α获得的重组蛋白。
进一步的,所述调控植物花粉管与柱头识别的硫氧还蛋白的制备方法,包括以下步骤:
①用正向引物和反向引物进行PCR扩增cDNA,PCR产物纯化后与载体pBS T连接,连接混合液转化大肠杆菌,得质粒pBSTrx;
②将质粒pBSTrx和质粒pET259用限制性内切酶EcoRV酶切,酶切片段用T4DNA连接酶连接,连接液转化入大肠杆菌,得质粒pETrx;
③将质粒pETrx转化BL21(DE3),所得转化子BL21(DE3)/pETrx纯化所得重组蛋白,制得氨基酸序列表如SEQ ID No.1或SEQ ID No.2所示的硫氧还蛋白;
所述正向引物的序列如SEQ ID No.5或SEQ ID No.6所示;
所述反向引物的序列如SEQ ID No.7或SEQ ID No.8所示;
使用序列SEQ ID No.5和SEQ ID No.7,制得的氨基酸序列表如SEQ ID No.1所示的硫氧还蛋白;
使用序列SEQ ID No.6和SEQ ID No.8,制得的氨基酸序列表如SEQ ID No.2所示的硫氧还蛋白。
进一步的,以拟南芥cDNA为模板。
进一步的,制备SEQ ID No.1所示硫氧还蛋白的插入突变体基因为gr1-1,制备SEQID No.2所示硫氧还蛋白的插入突变体基因为nrta-1。
进一步的,gr1-1插入到GR1的第三个内含子序列中,ntra-1插入到NTRA的第1个外显子序列中。
进一步的,GR1的基因序列表如SEQ ID No.3所示;NTRA的基因序列表如SEQ IDNo.4所示;
根据本发明实施例的第二方面,提供一种硫氧还蛋白于调控植物花粉管与柱头识别中的应用。
本发明实施例具有如下优点:本发明实施例提供一种调控植物花粉管与柱头识别的硫氧还蛋白及其制备方法,所述硫氧还蛋白的序列表如SEQ ID No.1或SEQ ID No.2所示。所述硫氧还蛋白为含有序列表SEQ ID No.1或SEQ ID No.2的真核重组表达质粒pETrx转化大肠杆菌DH5α获得的重组蛋白。通过用上述序列表的硫氧还蛋白能够有效提升花粉在柱头表面的水合和萌发以及花粉管在柱头表面的生长。
具体实施方式
下面将结合实施例对本发明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做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
本实施例提供一种调控植物花粉管与柱头识别的硫氧还蛋白,所述硫氧还蛋白的序列表如SEQ ID No.1或SEQ ID No.2所示。进一步的,所述硫氧还蛋白为含有序列表SEQID No.1或SEQ ID No.2的真核重组表达质粒pETrx转化大肠杆菌DH5α获得的重组蛋白。
实施例2
本实施例提供一种调控植物花粉管与柱头识别的硫氧还蛋白的制备方法,包括以下步骤:
①用正向引物和反向引物进行PCR扩增cDNA,PCR产物纯化后与载体pBS T连接,连接混合液转化大肠杆菌,得质粒pBSTrx;其中,以拟南芥cDNA为模板。
②将质粒pBSTrx和质粒pET259用限制性内切酶EcoRV酶切,酶切片段用T4DNA连接酶连接,连接液转化入大肠杆菌,得质粒pETrx;
③将质粒pETrx转化BL21(DE3),所得转化子BL21(DE3)/pETrx纯化所得重组蛋白,制得氨基酸序列表如SEQ ID No.1或SEQ ID No.2所示的硫氧还蛋白;
所述正向引物的序列如SEQ ID No.5或SEQ ID No.6所示;
所述反向引物的序列如SEQ ID No.7或SEQ ID No.8所示。
进一步的,制备SEQ ID No.1所示硫氧还蛋白的插入突变体基因为gr1-1,制备SEQID No.2所示硫氧还蛋白的插入突变体基因为nrta-1。
进一步的,gr1-1插入到GR1的第三个内含子序列中,ntra-1插入到NTRA的第1个外显子序列中。
进一步的,GR1的基因序列表如SEQ ID No.3所示;NTRA的基因序列表如SEQ IDNo.4所示;
根据本发明实施例的第二方面,提供一种硫氧还蛋白于调控植物花粉管与柱头识别中的应用。
实验一
为实现本申请技术方案的有效实施,申请人进行了以下实验,具体请参阅以下内容:
1)拟南芥gr1 ntra突变体的鉴定;
GR1基因的基因组序列包括16个外显子和15个内含子,NTRA基因的基因组序列包括2个外显子和1个内含子。从拟南芥T-DNA插入突变体库ABRC分别购买了这两个基因的T-DNA插入突变体,并分别命名为gr1-1和nrta-1。gr1-1插入到GR1的第三个内含子序列中,ntra-1插入到NTRA的第1个外显子序列中。
通过qRT-PCR分析发现,GR1和NTRA分别在gr1和ntra突变体中表达水平明显降低。
(2)gr1 ntra双突变的雄配子传递异常;
研究表明gr1和ntra单突变体没有表型,gr1 ntra ntrb导致不育,但是NTRB不在花粉中表达。从拟南芥突变体库购买了文章中用过的相关突变体进行验证,利用基因特异引物和T-DNA引物,通过PCR的方法鉴定发现:gr1/+雄配子传递效率降低(回交后代野生型和杂合体比例为60:32,大于理论值1:1),这可能是由于花粉数远大于胚珠数,使正常花粉完全可以使所有胚珠完成双受精,造成gr1/+没有表型;ntra/+雄配子传递效率正常(回交后代野生型和杂合体比例接近于1:1);ntra/+ntrb/-和ntra/-ntrb/+中雄配子传递效率也接近正常(回交后代野生型和杂合体的比例接近1:1);而gr1/+ntra/-或gr1/-ntra/+自交结果显示其后代植株中没有双纯合突变体,野生型与杂合体的分离比接近1:1,这表明gr1ntra的配子体是有缺陷的;为了研究gr1/+ntra/-和gr1/-ntra/+造成雄配子传递缺陷的原因,分别用这两种突变体与野生型进行了回交实验,发现当用杂合体做父本时gr1 ntra的传递效率为0,而杂合体做母本时其传递效率正常,这表明gr1 ntra双突变体的雄配子传递效率丧失。
(3)GR1和NTRA突变不影响花粉形态、细胞核发育和花粉活力;
为了确定gr1 ntra突变体中雄配子的功能障碍是否是由于花粉发育异常造成的,首先对野生型和突变体的花粉进行了扫描电镜的观察,结果发现,gr1/-、ntra/-、gr1/+ntra/-和gr1/-ntra/+的花粉外形与野生型相比没有异常;DAPI染色观察发现在gr1/-、ntra/-、gr1/+ntra/-和gr1/-ntra/+的花粉与野生型一样都包含两个生殖核和一个营养核,即突变体的花粉发育正常;亚历山大染色发现,gr1/-、ntra/-、gr1/+ntra/-和gr1/-ntra/+的花粉染色与野生型相似,表明突变体花粉活力正常。
(4)gr1 ntra花粉水合和体外萌发没有异常;
为了进一步确定突变的雄配子传递效率降低是否是由于花粉水合或者花粉萌发缺陷造成的,首先,用野生型的柱头,并利用限量授粉的方法进行了花粉水合实验,限量授粉5分钟后观察花粉水合情况发现gr1/-、ntra/-、gr1/+ntra/-和gr1/-ntra/+的花粉水合能力与野生型花粉相比没有差异;然后,利用体外花粉萌发实验检测了gr1/-、ntra/-、gr1/+ntra/-和gr1/-ntra/+花粉体外萌发(4小时)情况,发现突变体与野生型花粉管体外萌发率和花粉管形态上都是接近的。
(5)gr1 ntra的花粉不能完成受精;
为了进一步分析为什么突变的花粉配子传递效率为0,利用限量授粉的方法将WT、gr1/+ntra/-和gr1/-ntra/+的花粉授到野生型的柱头上,授粉后48小时统计膨大的胚珠与柱头上花粉的比例,经统计后发现gr1/+ntra/-和gr1/-ntra/+的花粉限量授到野生型的柱头上膨大的胚珠与柱头上相对应花粉的比例(分别是大约40%和48%)约是利用野生型花粉限量授粉到野生型的柱头上膨大的胚珠与柱头上花粉比例(约71%)的一半,这就证明gr1 ntra的花粉不能完成双受精。
(6)gr1 ntra花粉管在柱头表面停止生长;
为了探究gr1 ntra的花粉不能完成双受精的原因,利用限量授粉的方法观察了WT、gr1/-、ntra/-、gr1/+ntra/-和gr1/-ntra/+的花粉授到WT柱头上花粉管在柱头上的生长情况。限量授粉4小时后,发现WT花粉管已经进入花柱生长;gr1/-和ntra/-花粉管的生长情况与WT花粉管生长情况基本一致,但经统计发现gr1/-花粉管比WT花粉管多出10%在柱头异常生长的花粉管;但是,发现gr1/+ntra/-和gr1/-ntra/+各比WT多出约50%的花粉管不能穿过柱头。
以上结果表明,gr1 ntra花粉管在柱头表面停止生长,不能穿过柱头进入花柱等雌蕊组织。
(7)gr1 ntra花粉线粒体异常;
为了确定gr1 ntra花粉管在柱头表面停止生长,不能穿过柱头进入花柱的原因,又由于NTRA和GR1分别编码NADPH依赖的硫氧还蛋白还原酶和谷氧还蛋白还原酶,而氧化还原反应的场所一般是线粒体。因此,用Mito-tracker deep red对WT、gr1/-、ntra/-、gr1/+ntra/-和gr1/-ntra/+花粉线粒体进行了染色分析。
结果发现,花粉线粒体染色结果可分为线粒体正常、线粒体异常和线粒体显著异常三种情况。WT花粉正常线粒体约占总数的68%,异常线粒体占约12%,显著异常线粒体占约20%;gr1/-和ntra/-花粉正常线粒体约各占总数的15%和10%,异常线粒体各占约50%和55%,显著异常线粒体都约占35%;而gr1/+ntra/-和gr1/-ntra/+花粉正常线粒体约各占总数的15%和5%,异常线粒体各占约20%和10%,显著异常线粒体都约占65%和85%。
以上结果表明,gr1 ntra花粉线粒体出现严重异常,推测gr1 ntra花粉氧化还原状态及ROS水平与野生型相比会出现很大变化。
综上表明,GR1 NTRA在提升花粉在柱头表面的水合和萌发以及花粉管在柱头表面的生长过程中具有重要作用。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
序列表
<110> 中国林业科学研究院
<120> 一种调控植物花粉管与柱头识别的硫氧还蛋白及其制备方法
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 500
<212> Protein1
<213> 人工序列(Artificial Sequence)
<400> 1
markmlvdge idkvaadean athydfdlfv igagsggvra arfsanhgak vgicelpfhp 60
isseeiggvg gtcvirgcvp kkilvygaty ggeledakny gweinekvdf twkkllqkkt 120
deilrlnniy krllanaavk lyegegrvvg pnevevrqid gtkisytakh iliatgsraq 180
kpnipghela itsdealsle efpkraivlg ggyiavefas iwrgmgatvd lffrkelplr 240
gfddemralv arnlegrgvn lhpqtsltql tktdqgikvi sshgeefvad vvlfatgrsp 300
ntkrlnleav gveldqagav kvdeysrtni psiwavgdat nrinltpval meatcfanta 360
fggkptkaey snvacavfci pplavvglse eeaveqatgd ilvftsgfnp mkntisgrqe 420
ktlmklivde ksdkvigasm cgpdaaeimq giaialkcga tkaqfdstvg ihpssaeefv 480
tmrsvtrria hkpkpktnlz 500
<210> 2
<211> 379
<212> Protein1
<213> 人工序列(Artificial Sequence)
<400> 2
msqsrfiiks lfstaggfll gsalsnppsl atafssssss ssaaaavdme thktkvcivg 60
sgpaahtaai yasraelkpl lfegwmandi apggqltttt dvenfpgfpe gilgidivek 120
frkqserfgt tiftetvnkv dfsskpfklf tdsrtvlads viistgavak rlsftgsgeg 180
nggfwnrgis acavcdgaap ifrnkplvvi gggdsameea nfltkygskv yiihrrdtfr 240
askimqqral snpkieviwn savveaygde ngrvlgglkv knvvtgdvsd lkvsglffai 300
ghepatkfld gqleldedgy vvtkpgttkt svvgvfaagd vqdkkyrqai taagtgcmaa 360
ldaehylqei gsqegksdz 379
<210> 3
<211> 1500
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggcgagga agatgcttgt tgatggtgag attgataagg tggcggctga tgaagccaac 60
gcgacgcact atgattttga tttgtttgtc atcggtgccg ggagtggcgg tgttcgtgct 120
gctaggtttt cggctaatca tggcgctaag gttggtattt gtgagcttcc atttcaccct 180
attagctctg aggagattgg aggcgttggt ggaacctgtg ttatccgtgg ttgtgttcct 240
aaaaagattc tcgtctatgg agctacttac ggtggtgaac ttgaggatgc taaaaattat 300
gggtgggaaa taaatgagaa agtcgacttc acatggaaga agcttttgca aaagaagact 360
gatgagatac taagactgaa taatatctac aagcggttat tggcaaatgc tgcggtgaaa 420
ttgtatgaag gtgaaggaag agtagttggt cccaacgaag tggaggtgag acaaatagat 480
ggcacaaaaa taagttatac cgcaaagcac atattgattg ccactggcag tcgggcgcaa 540
aagcctaata ttcctggaca tgagctggct attacatctg atgaagcttt gagcttggaa 600
gaatttccca agcgtgctat agtgcttgga ggagggtata ttgctgtgga gtttgcatca 660
atatggcgtg gaatgggtgc tactgtagat ttattcttca ggaaggaact tccgctaagg 720
ggttttgatg acgaaatgag ggcactagtt gctagaaatc ttgaaggaag gggcgttaat 780
ctgcatccac aaacaagttt gactcagttg acaaaaacag accaggggat caaagtcata 840
tcgtcccatg gggaggaatt cgtggcagat gtcgtcctat ttgctactgg cagaagtcct 900
aataccaaaa gattgaattt agaagctgtt ggtgttgaac ttgatcaggc tggagctgtg 960
aaggttgacg agtattcacg aactaatata cctagcatat gggctgtagg agatgccaca 1020
aaccgaatta accttacacc tgttgcgtta atggaggcca cctgttttgc gaacactgct 1080
tttggtggaa agcctactaa agcagaatac agcaatgtcg cctgtgctgt attttgcata 1140
ccaccactag ctgtagtggg tctcagcgaa gaagaagcag tagaacaagc aaccggtgat 1200
attctggtct tcacctcagg ctttaatcca atgaagaaca ccatttctgg acgccaggaa 1260
aagacattga tgaagctaat agttgatgag aagagtgata aggttattgg agcatccatg 1320
tgcggtcctg atgcagctga gatcatgcag gggattgcaa ttgcgctcaa gtgtggagca 1380
accaaagcac aatttgatag cacggttggg atacatccat cttctgcaga ggaatttgtg 1440
acaatgcgca gtgtgaccag acgcattgcc cacaaaccca aacctaagac aaatctatga 1500
<210> 4
<211> 1737
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aaaaagattc tcgtctatgg agctacttac ggtggtgaac ttgaggatgc taaaaattat 300
gggtgggaaa taaatgagaa agtcgacttc acatggaaga agcttttgca aaagaagact 360
gatgagatac taagactgaa taatatctac aagcggttat tggcaaatgc tgcggtgaaa 420
ttgtatgaag gtgaaggaag agtagttggt cccaacgaag tggaggtgag acaaatagat 480
ggcacaaaaa taagttatac cgcaaagcac atattgattg ccactggcag tcgggcgcaa 540
aagcctaata ttcctggaca tgagctggct attacatctg atgaagcttt gagcttggaa 600
atgagccagt caagattcat tataaagtct ttatttagca cagcaggagg tttcttactt 660
ggatctgctc tctcaaatcc gccgtctcta gccaccgcgt tttcttcttc ttcctcgtcc 720
tcctccgccg ccgccgccgt cgacatggaa actcacaaaa ccaaggtttg catcgtcgga 780
agtggaccag cagcacacac ggcggcgatc tatgcatcga gagcggagct taagcctctt 840
ctcttcgaag gatggatggc taacgacatc gctcccggcg gtcaattaac tacaacaacc 900
gacgtcgaaa acttccctgg gttccctgaa ggtattctcg gtattgatat cgttgagaaa 960
ttcagaaaac aatcggagag atttggaact acgatcttca cggaaactgt taacaaagtt 1020
gatttctcat cgaaaccgtt taagctattc actgattcga gaactgttct cgctgattct 1080
gtaatcattt ctactggagc tgttgctaaa cgtcttagct tcactggatc tggtgaaggt 1140
aatggtggtt tttggaatcg tggtatctcc gcttgtgctg tttgcgacgg agctgctccg 1200
atttttagga ataagcctct tgtggttatt ggtggtggtg attcagctat ggaggaagcg 1260
aattttctga ctaagtatgg atctaaggtt tatattattc ataggaggga tacgtttagg 1320
gcgtctaaga ttatgcagca gagagctttg tctaacccta agattgaagt gatttggaac 1380
tctgccgtgg ttgaggcgta tggtgatgaa aatggacgtg ttcttggagg attgaaggtg 1440
aagaatgttg ttactgggga tgtttcagat ctgaaggtgt ctggattgtt ctttgctatt 1500
ggtcatgagc cagctacgaa gtttttggat gggcagcttg agcttgatga agatggttat 1560
gttgtgacca agccaggtac tactaagacg agcgtggttg gtgtatttgc tgctggagat 1620
gttcaagaca agaagtatag acaggccatc actgctgcag gaactgggtg catggcggca 1680
ttggatgcag agcattactt acaagagatt ggatctcagg agggtaagag tgattga 1737
<210> 5
<211> 19
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggcgagga agatgcttg 19
<210> 6
<211> 29
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgagccagt caagattcat tataaagtc 29
<210> 7
<211> 28
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 7
tcatagattt gtcttaggtt tgggtttg 28
<210> 8
<211> 23
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 8
tcaatcactc ttaccctcct gag 23
Claims (8)
1.一种调控植物花粉管与柱头识别的硫氧还蛋白,其特征在于,所述硫氧还蛋白的序列表如SEQ ID No.1或SEQ ID No.2所示。
2.根据权利要求1所述调控植物花粉管与柱头识别的硫氧还蛋白,其特征在于,所述硫氧还蛋白为含有序列表SEQ ID No.1或SEQ ID No.2的真核重组表达质粒pETrx转化大肠杆菌DH5α获得的重组蛋白。
3.根据权利要求1或2所述调控植物花粉管与柱头识别的硫氧还蛋白的制备方法,其特征在于,包括以下步骤:
①用引物F1和R1进行PCR扩增cDNA,PCR产物纯化后与载体pBS T连接,连接混合液转化大肠杆菌,得质粒pBSTrx;
②将质粒pBSTrx和质粒pET259用限制性内切酶EcoRV酶切,酶切片段用T4 DNA连接酶连接,连接液转化入大肠杆菌,得质粒pETrx;
③将质粒pETrx转化BL21(DE3),所得转化子BL21(DE3)/pETrx纯化所得重组蛋白,制得氨基酸序列表如SEQ ID No.1或SEQ ID No.2所示的硫氧还蛋白。
4.根据权利要求3所述的制备方法,其特征在于,以拟南芥cDNA为模板。
5.根据权利要求3所述的制备方法,其特征在于,制备SEQ ID No.1所示硫氧还蛋白的插入突变体基因为gr1-1,制备SEQ ID No.2所示硫氧还蛋白的插入突变体基因为nrta-1。
6.根据权利要求5所述的制备方法,其特征在于,gr1-1插入到GR1的第三个内含子序列中,ntra-1插入到NTRA的第1个外显子序列中。
7.根据权利要求6所述的制备方法,其特征在于,GR1的基因序列表如SEQ ID No.3所示;NTRA的基因序列表如SEQ ID No.4所示。
8.一种如权利要求1或2所述硫氧还蛋白于调控植物花粉管与柱头识别中的应用。
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