CN102876686A - 甘蔗psⅰ反应中心亚基ⅱ基因及其编码的蛋白质序列 - Google Patents
甘蔗psⅰ反应中心亚基ⅱ基因及其编码的蛋白质序列 Download PDFInfo
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- CN102876686A CN102876686A CN 201210338301 CN201210338301A CN102876686A CN 102876686 A CN102876686 A CN 102876686A CN 201210338301 CN201210338301 CN 201210338301 CN 201210338301 A CN201210338301 A CN 201210338301A CN 102876686 A CN102876686 A CN 102876686A
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Abstract
本发明公开了一种甘蔗PSⅠ反应中心亚基Ⅱ基因及其编码的蛋白质序列，在对所有植物PSⅠ反应中心亚基Ⅱ基因全长序列进行进化分析的基础上，以同一家族的PSⅠ反应中心亚基Ⅱ基因序列的保守区设计引物，从甘蔗幼嫩叶片提取总RNA，并经反转录，用常规PCR法扩增，结合3’RACE PCR技术克隆到甘蔗PSⅠ反应中心亚基Ⅱ基因的全长cDNA序列，经VectorNTI软件分析，该基因ORF为603bp，编码200个氨基酸。不仅为研究甘蔗PSⅠ反应中心亚基Ⅱ基因的转录和表达机制，进一步探讨PSⅠ反应中心亚基Ⅱ在PSⅠ作用机理奠定基础，而且通过氨基酸序列可以获得具有生物活性的纯化蛋白，为研究PSⅠ反应中心亚基Ⅱ类酶的生物学功能提供基础，此外，提高转基因等技术，可用于农作物改良。
Description
技术领域
本发明属于生物工程技术领域，涉及一种甘蔗PSⅠ反应中心亚基Ⅱ基因及其编码的蛋白质序列。
背景技术
光合作用是植物特有属性，通过光合作用合成有机物是地球上生物最重要的物质和能量来源，产生的氧气是维持大气平衡的重要基础。阐明光合作用机理，提高农作物光合作用效率，维持大气平衡，降低温室效应具有重要意义。高等植物有2种光系统：光系统Ⅰ(PSⅠ)和光系统Ⅱ(PSⅡ)，每一系统均由捕光复合物和反应中心所组成。PSⅠ是整合于光合膜上的有多个蛋白亚基组成的色素蛋白复合物，参与光氧化还原反应和光合电子传递链中，催化电子从囊腔侧的质体兰素(plastocyanin，PC)经过一系列电子传递体到向基质侧的铁氧还蛋白(ferredoxin，Fd)过程。
PSⅠ由PSⅠ核心复合物和捕光色素蛋白复合物LHCⅠ组成。Jensen认为PSⅠ核心复合物至少具有14个亚基（分别为PsaA，PsaAB，……，PsaN），其编码基因位于细胞核或者叶绿体上，功能各不相同，可作为反应中心蛋白（如PsaA和PsaAB）、结合质蓝素（如PsaF）、参与循环电子传递（PsaE）、与LHCI连接（PsaH）、Fe-S脱辅基蛋白（PsaC）、结合铁氧还蛋白（PsaD）等，其中还有不少未知功能蛋白或者功能有争议的蛋白。PsaG、PsaI、PsaJ、PsaM、PsaK、PsaL等功能未知。拟南芥中PSⅠ核心复合物亚基PsaG、PsaF、PsaJ和PsaK均分别与LHCA1、LHCA4、LHCA2、LHCA3结合。PSⅠ中多肽组分的结构及功能、PSⅠ电子传递反应、天线色素系统中激发能的传递以及激发能到反应中心的陷落是目前对PSI的结构和功能的研究主要方向。光照和温度是影响光合作用的两个重要的因素，通过研究光合器官在不同光照和温度下的响应机制，有助于阐明光合作用的内在机理。
PasD在蓝细菌中由139-144个氨基酸残基组成，分子量15.5kDa，而在高等植物中大约有23个氨基酸残基组成的N端延伸片段，其分子量约18kDa。林文雄应用差异蛋白质技术从水稻中分离出一个PsaD蛋白，发现它可促进PSⅠ反应中心的电子聚合，增强PSⅠ与铁氧化蛋白有效结合。从其3D结构预测，铁、镁、钙离子是该蛋白不可或缺的一部分，而这些离子可以明显促进PSⅠ中电子的传递。
本发明甘蔗PSⅠ反应中心亚基Ⅱ基因（photosystem I reaction center subunit II）属于psaD基因，所编码的蛋白质属于PsaD蛋白。到目前还没有从甘蔗上克隆到psaD基因，对其功能还不清楚。在本发明基础上，构建psaD基因的真核表达载体，将其转入烟草和甘蔗等，可以对psaD基因的功能进行分析，有助于进一步揭示psaD基因在PSⅠ的作用，进行农作物品种改良。通过构建原核表达载体，可以分离纯化PsaD蛋白，研究其酶学特性，阐明PsaD蛋白组分的性质。
发明内容
本发明的目的：提供一种甘蔗PSⅠ反应中心亚基Ⅱ基因及其编码的蛋白质序列，为进一步研究甘蔗PSⅠ反应中心亚基Ⅱ的功能，以及应用甘蔗PSⅠ反应中心亚基Ⅱ改良农作物品种奠定良好基础。
本发明所采取的技术方案如下：
一种甘蔗PSⅠ反应中心亚基Ⅱ基因及其编码蛋白质序列，是在对所有植物PSⅠ反应中心亚基Ⅱ全长序列进行进化分析的基础上，比对同一家族的PSⅠ反应中心亚基Ⅱ的cDNA序列，并设计扩增PSⅠ反应中心亚基Ⅱ酶基因的引物。从甘蔗幼嫩叶片提取总RNA，并经反转录，用RT-PCR法扩增PSⅠ反应中心亚基Ⅱ类酶基因片段。利用3′RACE PCR技术扩增到PSⅠ反应中心亚基Ⅱ酶基因全长cDNA序列，总长914bp。经VectorNTI软件分析，该序列包含一个完整的读码框，ORF为603bp，编码200个氨基酸，起始密码子(ATG)位于转录起始位点后81bp处，终止密码子(TAG)后还有一段231bp的非编码序列，并带有真核生物典型的polyA尾巴。
本发明所获得的甘蔗PSⅠ反应中心亚基Ⅱ的3′端序列，是用以下核苷酸序列为上游引物，与3′-Full RACEKit(TAKARA)提供的引物SoGZF1：（5’-ggccacgcgtcgactagtac-3′）扩增获得。
以上所述的甘蔗PSⅠ反应中心亚基Ⅱ，其特征在于：它具有序列表中SEQID NO.1的DNA序列，且它的编码区从第81位核苷酸到683位核苷酸。
以下是本发明所获得的甘蔗PSⅠ反应中心亚基Ⅱ酶的全长cDNA序列：
ggccacgcgtcgactagtactctcctcttgctcgattcgcttgctcgatccttcctagttccttccaagaagcagcgccc
gccatggccactcaagcctccgccgccacgcgccacctgctggccgccgcctgggcgaagccacggcagctgagctcgcagctcgcgatgccgccgccgtcgtcctcccgcgggccggccccgctccgcgcgtccgccgaggaggcggtgaccaaggaggcgcccaaggggttcgtgcccccgcagctggaccccaacacgccgtcgccgatcttcggcggcagcacgggcgggctgcttcgcaaggcgcaggtggaggagttctacgtcatcacgtggacgtccccgaaggagcaggtgttcgagatgcccacgggcggcgccgccatcatgcgggaggggcccaacctcctgaagctggcgcgcaaggagcagtgcctggcgctcggcaccaggctgcgctccaagtacaagatcaactaccagttctaccgcgtcttccccaacggcgaggtgcagtacctccaccccaaggacggcgtctacccggagaaggtcaacgccggcaggcagggcgtcggccagaacttccgcagcatcggcaagaacgtcagccccatcgaggtcaagttcaccggcaagaacaccttcgacatc
tccatcgcatgcatcacgtgcggccggcgacccaaattgaaggacttcttcttatatgaattataagttagcggtgctgccatgcatggtttgatagatgatccatgtgtaattttattatcaagttttcttggtgaccgtggaggcgcagcgtgggattatatgtacatgttcatttgctcgtcataacgttgcttatttgcataccgttaaaaaaaaaaaaaaaaaaaa。
甘蔗PSⅠ反应中心亚基Ⅱ编码的蛋白质，是具有序列表中SEQID NO.2氨基酸残基序列的蛋白质，或者是将SEQID NO.2的氨基酸残基序列经过一个以上氨基酸残基的取代和/或缺失戓添加且具有与SEQID NO.2的氨基酸残基序列相同活性的衍生蛋白质。
以下是本发明所获得的甘蔗PSⅠ反应中心亚基Ⅱ所编码蛋白的氨基酸序列：
MAMATQASAATRHLLAAAWAKPRQLSSQLAMPPPSSSRGPAPLRASAEEAVTKEAPKGFVPPQLDPNTPSPIFGGSTGGLLRKAQVEEFYVITWTSPKEQVFEMPTGGAAIMREGPNLLKLARKEQCLALGTRLRSKYKINYQFYRVFPNGEVQYLHPKDGVYPEKVNAGRQGVGQNFRSIGKNVSPIEVKFTGKNTFDI。
本发明的优点及其效果：
本发明的甘蔗PSⅠ反应中心亚基Ⅱ基因及其编码的蛋白质序列，不仅为研究甘蔗PSⅠ的作用，提高电子传递和光合作用速率，而且为进一步研究PSⅠ反应中心亚基Ⅱ的转录和表达机制，探讨PSⅠ反应中心亚基Ⅱ在PSⅠ中所起到的作用、生物学功能及利用该基因进行甘蔗品种改良奠定基础。
附图说明
图1：甘蔗PSⅠ反应中心亚基Ⅱ的全长cDNA序列；
图2：甘蔗PSⅠ反应中心亚基Ⅱ所编码蛋白质的氨基酸序列。
具体实施方式
实施例
1、植物PSⅠ反应中心亚基Ⅱ基因进化分析：
用“photosystem I reaction center subunit II”搜索NCBI网站(http://www.ncbi.nlm.nih.gov)非冗余氨基酸数据库，获得所有植物光合系统Ⅰ反应中心亚基Ⅱ基因序列。对所获得的所有序列进行分析，除去重复的序列，共得到PSⅠ反应中心亚基Ⅱ序列50条，其中全长序列8条。用ClustalX对所有全长PSⅠ反应中心亚基Ⅱ的氨基酸序列进行比对，并将比对结果保存为PHYLIP格式；然后用PHYLIP软件包的Seqboot,Protdist,Neighbor和Consense程序进行进化分析，将植物所有全长PSⅠ反应中心亚基Ⅱ基因分为相应的家族。
2、甘蔗总RNA的提取：
(1)取新鲜甘蔗幼嫩叶片，在液氮中充分研磨成粉末状，称取100mg，放入用液氮预冷过的1.5ml离心管中，加入1ml Trizol-A+提取液(天根生化科技有限公司)。
(2)将样品充分混匀，室温下放置5min，使得核酸蛋白复合物完全分离。
(3)4℃，14,000g，离心10min。
(4)吸取上清液于另一个1.5ml离心管中，加入氯仿体积为上清液体积的1/5，充分振荡混匀15sec，室温放置2-3min。
(5)4℃，14,000g，离心15min。
(6)重复步骤4和5一次。
(7)吸取上层水相溶液于另一个1.5ml离心管中，加入等体积预冷的异丙醇，混匀10min，冰上放置25min。
(8)4℃，14,000g，离心10min后，在管侧和管底形成白色胶状沉淀。
(9)去上清液，留沉淀。加入1ml 75%乙醇洗涤沉淀2遍(每次4℃，2,300g，离心3min）。
(10)倒出液体，室温放置3-5min；加入30-50μl DEPC处理水，充分溶解后于-70℃保存。
(11)取1μl RNA样品用1%琼脂糖凝胶进行电泳检测，另取1μl RNA样品测浓度。
3、RT-PCR（反转录PCR）：
(1)准备一个0.2ml的离心管，加入以下成分：
甘蔗总RNA: 0.1-5μg
oligo(dT)18引物(0.5μg/μl)： 1μl
加入DEPC-treated water到总体积： 12μl。
(2)轻微混匀,简短离心之后，65°C温育5min，放置冰上冷却。
(3)按要求加入以下成分：
5×Reaction Buffer： 4μl
RiboLockTM RNase Inhibitor(20U/μl)： 1μl
10mM dNTP Mix： 2μl
ReverAidTM M-MuLV Reverse Tranxcriptase： 1μl
总体积为： 20μl。
(4)轻微混匀，简短离心。
(5)42°C温育60min。
(6)70°C温育5min终止反应，-20°C保存备用。
4、PSⅠ反应中心亚基Ⅱ酶3′-RACE克隆：
根据不同PSⅠ反应中心亚基Ⅱ基因核苷酸序列的同源性，在核苷酸序列保守区设计一条特异的上游引物。
SoGZF1：（5′-ggccacgcgtcgactagtac-3′）；
3′-Full RACEKit(TAKARA)提供的锚定引物：
SoGZR1：（5′-ggccacgcgtcgactagtac-3′）
GZS：(5′-ggccacgcgtcgactagtactttttttttttttttttt-3′)。
提取甘蔗幼嫩叶总RNA，按照上述RT-PCR方法，以GZS核苷酸序列为反转录引物，逆转录合成cDNA；用SoGZF1和SoGZⅠR1引物进行PCR扩增。产物经胶回收纯化后克隆到T载体并测序。测序结果表明，产物长度914bp。
5、甘蔗PSⅠ反应中心亚基Ⅱ酶全长序列分析：
对其所编码的氨基酸在NCBI上进行“protein blast”比对分析，发现914bp序列已经包含PSⅠ反应中心亚基Ⅱ酶基因全长cDNA序列。经VectorNTI软件分析，该序列的ORF为603bp，编码200个氨基酸。1-80bp为5′端非编码区，起始密码子(ATG)位于转录起始位点后81bp处，终止密码子(TAA)位于转录起始位点后683bp处，其后还有一段231bp的非编码序列，并带有真核生物典型的polyA尾巴。
下表为甘蔗PSⅠ反应中心亚基Ⅱ酶序列表。
序列表
<110>广西大学
<120>甘蔗光合系统Ⅰ反应中心亚基Ⅱ基因及其编码的蛋白质序列
<160>2
<210>1
<211>914
<212>DNA
<213>甘蔗（Saccharum officinarum）
<220>
<221>CDS
<222>(81)...(683)
<220>
<221>5’UTP
<222>(1)...(80)
<220>
<221>3’UTP
<222>(684)...(914)
<400>1 GGCCACGCGT CGACTAGTAC TCTCCTCTTG 20
CTCGATTCGC TTGCTCGATC CTTCCTAGTT CCTTCCAAGA AGCAGCGCCC 80
ATG GCC ATG GCC ACT CAA GCC TCC GCC GCC ACG CGC CAC CTG CTG 125
Met Ala Met Ala Thr Gln Ala Ser Ala Ala Thr Arg His Leu Leu
5 10 15
GCC GCC GCC TGG GCG AAG CCA CGG CAG CTG AGC TCG CAG CTC GCG 170
Ala Ala Ala Trp Ala Lys Pro Arg Gln Leu Ser Ser Gln Leu Ala
20 25 30
ATG CCG CCG CCG TCG TCC TCC CGC GGG CCG GCC CCG CTC CGC GCG 215
Met Pro Pro Pro Ser Ser Ser Arg Gly Pro Ala Pro Leu Arg Ala
35 40 45
TCC GCC GAG GAG GCG GTG ACC AAG GAG GCG CCC AAG GGG TTC GTG 260
Ser Ala Glu Glu Ala Val Thr Lys Glu Ala Pro Lys Gly Phe Val
50 55 60
CCC CCG CAG CTG GAC CCC AAC ACG CCG TCG CCG ATC TTC GGC GGC 305
Pro Pro Gln Leu Asp Pro Asn Thr Pro Ser Pro Ile Phe Gly Gly
65 70 75
AGC ACG GGC GGG CTG CTT CGC AAG GCG CAG GTG GAG GAG TTC TAC 350
Ser Thr Gly Gly Leu Leu Arg Lys Ala Gln Val Glu Glu Phe Tyr
80 85 90
GTC ATC ACG TGG ACG TCC CCG AAG GAG CAG GTG TTC GAG ATG CCC 395
Val Ile Thr Trp Thr Ser Pro Lys Glu Gln Val Phe Glu Met Pro
95 100 105
ACG GGC GGC GCC GCC ATC ATG CGG GAG GGG CCC AAC CTC CTG AAG 440
Thr Gly Gly Ala Ala Ile Met Arg Glu Gly Pro Asn Leu Leu Lys
110 115 120
CTG GCG CGC AAG GAG CAG TGC CTG GCG CTC GGC ACC AGG CTG CGC 485
Leu Ala Arg Lys Glu Gln Cys Leu Ala Leu Gly Thr Arg Leu Arg
125 130 135
TCC AAG TAC AAG ATC AAC TAC CAG TTC TAC CGC GTC TTC CCC AAC 530
Ser Lys Tyr Lys Ile Asn Tyr Gln Phe Tyr Arg Val Phe Pro Asn
140 145 150
GGC GAG GTG CAG TAC CTC CAC CCC AAG GAC GGC GTC TAC CCG GAG 575
Gly Glu Val Gln Tyr Leu His Pro Lys Asp Gly Val Tyr Pro Glu
155 160 165
AAG GTC AAC GCC GGC AGG CAG GGC GTC GGC CAG AAC TTC CGC AGC 620
Lys Val Asn Ala Gly Arg Gln Gly Val Gly Gln Asn Phe Arg Ser
170 175 180
ATC GGC AAG AAC GTC AGC CCC ATC GAG GTC AAG TTC ACC GGC AAG 665
Ile Gly Lys Asn Val Ser Pro Ile Glu Val Lys Phe Thr Gly Lys
185 190 195
AAC ACC TTC GAC ATC 680
Asn Thr Phe Asp Ile
200
TAATCCATCG CATGCATCAC GTGCGGCCGG CGACCCAAAT TGAAGGACTT CTTCTTATAT 740
GAATTATAAG TTAGCGGTGC TGCCATGCAT GGTTTGATAG ATGATCCATG TGTAATTTTA 800
TTATCAAGTT TTCTTGGTGA CCGTGGAGGC GCAGCGTGGG ATTATATGTA CATGTTCATT 860
TGCTCGTCAT AACGTTGCTT ATTTGCATAC CGTTAAAAAA AAAAAAAAAA AAAA 914
<210>2
<211>200
<212>PRT
<213>甘蔗（Saccharum officinarum）
<400>2
Met Ala Met Ala Thr Gln Ala Ser Ala Ala Thr Arg His Leu Leu
5 10 15
Ala Ala Ala Trp Ala Lys Pro Arg Gln Leu Ser Ser Gln Leu Ala
20 25 30
Met Pro Pro Pro Ser Ser Ser Arg Gly Pro Ala Pro Leu Arg Ala
35 40 45
Ser Ala Glu Glu Ala Val Thr Lys Glu Ala Pro Lys Gly Phe Val
50 55 60
Pro Pro Gln Leu Asp Pro Asn Thr Pro Ser Pro Ile Phe Gly Gly
65 70 75
Ser Thr Gly Gly Leu Leu Arg Lys Ala Gln Val Glu Glu Phe Tyr
80 85 90
Val Ile Thr Trp Thr Ser Pro Lys Glu Gln Val Phe Glu Met Pro
95 100 105
Thr Gly Gly Ala Ala Ile Met Arg Glu Gly Pro Asn Leu Leu Lys
110 115 120
Leu Ala Arg Lys Glu Gln Cys Leu Ala Leu Gly Thr Arg Leu Arg
125 130 135
Ser Lys Tyr Lys Ile Asn Tyr Gln Phe Tyr Arg Val Phe Pro Asn
140 145 150
Gly Glu Val Gln Tyr Leu His Pro Lys Asp Gly Val Tyr Pro Glu
155 160 165
Lys Val Asn Ala Gly Arg Gln Gly Val Gly Gln Asn Phe Arg Ser
170 175 180
Ile Gly Lys Asn Val Ser Pro Ile Glu Val Lys Phe Thr Gly Lys
185 190 195
Asn Thr Phe Asp Ile
200
Claims (2)
1.甘蔗PSⅠ反应中心亚基Ⅱ基因及其编码的蛋白质序列，其特征在于：
a、甘蔗PSⅠ反应中心亚基Ⅱ基因,它具有SEQ ID NO.1的DNA序列，且它的编码区从第81位核苷酸到683位核苷酸；
SEQ ID NO.1甘蔗PSⅠ反应中心亚基Ⅱ基因的全长cDNA序列是：
ggccacgcgtcgactagtactctcctcttgctcgattcgcttgctcgatccttcctagttccttccaagaagcagcgccc
gccatggccactcaagcctccgccgccacgcgccacctgctggccgccgcctgggcgaagccacggcagctgagctcgcagctcgcgatgccgccgccgtcgtcctcccgcgggccggccccgctccgcgcgtccgccgaggaggcggtgaccaaggaggcgcccaaggggttcgtgcccccgcagctggaccccaacacgccgtcgccgatcttcggcggcagcacgggcgggctgcttcgcaaggcgcaggtggaggagttctacgtcatcacgtggacgtccccgaaggagcaggtgttcgagatgcccacgggcggcgccgccatcatgcgggaggggcccaacctcctgaagctggcgcgcaaggagcagtgcctggcgctcggcaccaggctgcgctccaagtacaagatcaactaccagttctaccgcgtcttccccaacggcgaggtgcagtacctccaccccaaggacggcgtctacccggagaaggtcaacgccggcaggcagggcgtcggccagaacttccgcagcatcggcaagaacgtcagccccatcgaggtcaagttcaccggcaagaacaccttcgacatc
tccatcgcatgcatcacgtgcggccggcgacccaaattgaaggacttcttcttatatgaattataagttagcggtgctgccatgcatggtttgatagatgatccatgtgtaattttattatcaagttttcttggtgaccgtggaggcgcagcgtgggattatatgtacatgttcatttgctcgtcataacgttgcttatttgcataccgttaaaaaaaaaaaaaaaaaaaa；
b、甘蔗PSⅠ反应中心亚基Ⅱ基因编码的蛋白质，是具有SEQ ID NO.2序列表中氨基酸残基序列的蛋白质；
SEQ ID NO.2甘蔗PSⅠ反应中心亚基Ⅱ酶基因所编码蛋白的氨基酸序列是：
MAMATQASAATRHLLAAAWAKPRQLSSQLAMPPPSSSRGPAPLRASAEEAVTKEAPKGFVPPQLDPNTPSPIFGGSTGGLLRKAQVEEFYVITWTSPKEQVFEMPTGGAAIMREGPNLLKLARKEQCLALGTRLRSKYKINYQFYRVFPNGEVQYLHPKDGVYPEKVNAGRQGVGQNFRSIGKNVSPIEVKFTGKNTFDI。
2.根据权利要求1所述的甘蔗PSⅠ反应中心亚基Ⅱ基因及其编码蛋白质序列，其特征在于：所述的甘蔗PSⅠ反应中心亚基Ⅱ基因的全长cDNA序列，是用以下核苷酸序列为上游引物与3′-Full RACE Kit(TAKARA)提供的锚定引物扩增获得：
a、用于扩增甘蔗PSⅠ反应中心亚基Ⅱ酶基因全长上游引物为：
SoGZF1:（5′-ggccacgcgtcgactagtac-3′）。
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