CN108130334A - 柳枝稷s-腺苷甲硫氨酸合成酶基因sams1调控木质素合成的应用 - Google Patents

柳枝稷s-腺苷甲硫氨酸合成酶基因sams1调控木质素合成的应用 Download PDF

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CN108130334A
CN108130334A CN201711439616.9A CN201711439616A CN108130334A CN 108130334 A CN108130334 A CN 108130334A CN 201711439616 A CN201711439616 A CN 201711439616A CN 108130334 A CN108130334 A CN 108130334A
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pvsams1
lignin
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付春祥
熊王丹
吴振映
刘雨辰
齐天雄
刘文文
刘金丽
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Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
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Abstract

本发明公开了柳枝稷S‑腺苷甲硫氨酸合成酶基因SAMS1在改变木质素含量，进而提高糖化效率方面的应用，属于植物基因工程技术领域。其主要内容包括：柳枝稷SAMS1基因克隆和序列测定；构建PvSAMS1基因的干扰表达载体（pANIC8B‑PvSAMS1‑RNAi）；采用农杆菌介导法转化柳枝稷愈伤，将靶标基因的干涉片段转入低地型Alamo野生型柳枝稷中，获得木质素总量降低的转基因植株。所获得转基因植株中PvSAMS1表达量显著性降低，木质素含量显著降低，糖化效率显著性增加。本发明所鉴定的柳枝稷SAMS1对木质素代谢的调控，能够为今后分子育种提供新的靶标。

Description

柳枝稷S-腺苷甲硫氨酸合成酶基因SAMS1调控木质素合成的 应用
技术领域
本发明属于植物基因工程技术领域，具体涉及柳枝稷SAMS1表达水平的改造，改变木质素含量，提高柳枝稷糖化效率。
背景技术
柳枝稷（Panicum virgatum L.）属禾本科黍属，为多年生C4草本植物。它是第二代模式能源植物，也是重要的牧草资源。植物细胞壁主要由木质素、纤维素和半纤维素组成，它们是决定生物质能源转化效率和牧草品质的重要因素。柳枝稷作为生物质能源，解析其木质素代谢途径相关的调控机制，提高糖化效率，具有重要的现实指导意义。
木质素的合成途径中其单体的甲基化过程由咖啡酰辅酶A氧甲基转移酶和咖啡酸氧甲基转移酶催化生成的，其甲基化过程需要一碳代谢为其提供甲基。一碳代谢主要包括四氢叶酸循环和甲硫氨酸循环。S-腺苷甲硫氨酸合成酶（S-adenosyl methioninesynthase, SAMS）是甲硫氨酸循环的关键酶之一，它以甲硫氨酸为底物生成S-腺苷甲硫氨酸（S-adenosyl methionine, SAM）。因此，SAM是一碳单位的直接供体，可以为木质素单体甲基化提供甲基供体。拟南芥中mto3突变体中SAMS基因的突变，导致植物体内SAM大量积累，木质素含量显著性降低（Shen et al, High free-methionine and decreased lignincontent result from a mutation in the ArabidopsisS-adenosyl-L-methioninesynthetase 3 gene. Plant Journal, 2002, 29(3): 371-380）。但是甲硫氨酸途径对木质素的调控机制，目前尚未有相关报道。
本发明主要针对柳枝稷的SAMS，通过干扰沉默技术降低PvSAMS1在体内的表达水平，降低甲基供给水平，最终实现对柳枝稷木质素单体合成的调控。本研究不仅为深入理解柳枝稷及其它禾本科植物木质素代谢调控机制提供理论基础，也为定向分子遗传育种提供现实指导意义。
发明内容
本发明的第一个目的是提供一种柳枝稷一碳代谢合成酶PvSAMS1的编码基因；其核苷酸序列和氨基酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
本发明的第二个目的是提供一种针对PvSAMS1保守区域，其核苷酸序列如SEQ IDNO.3所示。利用此片段构建干涉载体，降低柳枝稷体内PvSAMS1的表达水平。
本发明的第三个目的是提供柳枝稷PvSAMS1在调控柳枝稷木质素合成及可发酵糖产量方面的应用。
所述柳枝稷PvSAMS1的RNAi片段，基于Gateway技术重组整合入表达载体pANIC8B中；采用农杆菌EHA105介导的遗传转化方法，将目的片段导入柳枝稷胚性愈伤细胞中，通过潮霉素抗性筛选，获得抗性再生植株，并通过PCR分析最终确定阳性转基因植株；木质素含量及糖化效率测定结果表明，PvSAMS1能够显著降低柳枝稷木质素含量，提高糖化效率。
本发明的核心特点和发明理念包括：
1、本发明利用基因工程的手段，通过干扰沉默技术降低PvSAMS1在柳枝稷体内的表达水平，能够在短期内获得显著效果；
2、本发明从调控一碳代谢途径的基因着手，分析柳枝稷木质素合成途径的调控机制，进一步深入解析了单子叶植物中木质素调控的机制。
本发明的有益效果如下：
1、本发明中获得的PvSAMS1基因是调控柳枝稷木质素单体组分的关键基因，这对于通过分子育种手段获得高转化效率的植株具有重要意义；
2、本发明中对PvSAMS1进行分子调控，能够显著增加柳枝稷的糖化效率，对于能源植物和禾本科牧草的生物量和糖化效率的遗传改良具有重要的参考意义；
3、本发明中所产生的遗传改良植物能够整合到常规育种项目，从而为能源植物和禾本科牧草作物的品种培育提供新的种质资源。
附图说明
图1 柳枝稷SAMS系统进化树
图2 柳枝稷PvSAMS1-RNAi表达载体简图；
图3 转pANIC8B-PvSAMS1-RNAi基因植株的PCR鉴定。M表示DNA Marker，ctrl plant表示转pANIC8B空载体的柳枝稷植株，T1-T7分别表示转基因植株的hph探针检测。hph为pANIC8B载体上携带的植物抗性筛选标记基因。
图4 转pANIC8B-PvSAMS1-RNAi植株的qRT-PCR分析。ctrl plant表示转pANIC8B空载体的柳枝稷植株，T1-T7分别表示转基因植株的hph探针检测。
图5 转基因植株木质素总量的测定。ctrl plant表示转pANIC8B空载体的柳枝稷植株。
图6 转基因植株糖化效率的测定。ctrl plant表示转pANIC8B空载体的柳枝稷植株。
具体实施方式
下面以针对PvSAMS1-RNAi降低柳枝稷木质素总量、增加糖化效率为实施例对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定发明的范围。下述实施例中所用的材料、试剂、双元载体和农杆菌等，如无特殊说明，均可从公司通过商业途径购到，所述的MS基本培养基购自PhytoTechnology Laboratories（货号M519）。
实施例1：PvSAMS1基因的克隆与序列测定
首先，我们根据水稻SAMS相关研究为研究背景（Li et al., Knockdown of SAMSgenes encoding S-adenosyl -L-methionine synthetases causes methylationalterations of DNAs and histones and leads to late flowering in rice. 2011, JPlant Physiol, 168:1837-1843），全基因组检索柳枝稷数据库，获得柳枝稷SAMS候选基因，并构建系统进化树（如图1所示）。然后，我们取低地型柳枝稷Alamo的嫩茎部位，用TriZol Reagent（Invitrogen公司，货号15596026）提取嫩茎总RNA，使用琼脂糖凝胶电泳和核酸分析仪（NanoDrop）检测总RNA的含量和纯度，取2.0 μg总RNA做逆转录反应，所采用的逆转录酶为M-MLV（Promega公司，货号M1701），逆转录反应的步骤参考该逆转录酶的使用说明。以逆转录反应合成的第一链cDNA为模板，使用引物：
F1: 5’CTGAGACGAGCAATACCCATC3’
R1: 5’GCCTTAAGCAGAAGGCTTCTC3’
进行常规PCR扩增；PCR反应体系为：2 μL cDNA，5 μL 10×Buffer，4 μL dNTP（2.5mM），正/反向引物（10 μM）各1 μL，0.5 μL Taq酶（5 U/μL）和36.5 μL ddH2O。在冰上加样后混匀。PCR反应条件为：94 oC 5 min；94 oC 30 sec，56 oC 30 sec；72 oC 90 sec，32个循环；72 oC 10 min。PCR扩增获得约1.4 kb的片段。采用琼脂糖凝胶电泳回收该片段后，连接pMD-19T载体（TaKaRa公司，货号6011）上，连接反应：4.5 μL PCR产物，0.5 μL pMD-19T载体和5 μL Solution I，16 oC连接2 h。连接产物用热激法转化大肠杆菌DH5α感受态细胞，加800 μL液体LB培养基，复苏1 hr，涂于氨苄抗生素的LB平板上（已涂布X-gal/IPTG），37 oC培养14 hr。挑选白色单克隆菌落，在含氨苄抗生素的液体LB培养基中扩增培养，测序。经测序分析表明，该序列含有一个完整的开放阅读框，全长1191个碱基，序列如SEQ ID NO.1所示，其编码蛋白含有396个氨基酸残基，序列如SEQ ID NO.2所示。
实施例2：PvSAMS1基因干扰沉默表达载体构建和遗传转化
本发明所用的干扰沉默表达载体是pANIC8B。通过PCR反应克隆获得PvSAMS1干扰片段（SEQ ID NO.3），使用引物：
F2: 5’TGGACCTCATGGTGATGCTGG3’ R2: 5’GCAGAAGGCTTCTCCCACTTG3’
通过同源重组in fusion的方法将目的片段连到改造后的入门载体pENTR上，碱裂解法提取测序正确的重组菌株质粒，EcoR V核酸内切酶37 oC酶切1 h，最后通过Gateway技术将目的片段重组到pANIC8B载体上（图2）。重组反应为：100 ng酶切回收片段，50 ng pANIC8B载体质粒，1 μL LR酶（Invitrogen，货号11791020），然后用ddH2O补足至10 μL。16 oC连接过夜。取5 μL连接产物，用热激法转化大肠杆菌DH5α感受态细胞，加800 μL液体LB培养基，复苏1 hr，涂于卡那霉素抗性的LB平板上（已涂布X-gal/IPTG），37 oC培养14 hr。挑选白色单克隆菌落，在卡那霉素抗性的LB培养基中扩增培养，测序。碱裂解法提取测序正确的重组菌株质粒，电击法转化农杆菌AGL1感受态细胞，涂布于含卡那霉素和利福平的LB平板上，28oC，暗培养2天，使用上述引物检测阳性单克隆，在卡那霉素和利福平抗性的液体LB培养基中扩增培养，用于柳枝稷胚性愈伤的遗传转化。
采用农杆菌介导的柳枝稷胚性愈伤的遗传转化方法（Xi et al, Agrobacterium-mediated transformation of switchgrass and inheritance of the transgenes.2009, Bioenergy Research, 2: 275-283）将pANIC8B-PvSAMS1-RNAi导入低地型野生型柳枝稷，获得抗性苗，经PCR鉴定扩增hph基因，最终确定阳性转基因株系（图3）。
实施例3：转基因植株分子鉴定
取上述鉴定转基因阳性植株嫩茎组织，使用TriZol（Invitrogen公司，货号15596026）法提取总RNA，逆转录酶（Promega公司，货号M1701）合成第一链cDNA，以柳枝稷Ubiquitin基因作为内参基因，分别采用引物PvSAMS1-F3/R3检测阳性转基因植株PvSAMS1基因的表达情况（图4），引物序列如下：
PvUbiq-F: 5’-TTCGTGGTGGCCAGTAAG-3’
PvUbiq-R: 5’-AGAGACCAGAAGACCCAGGTACAG-3’
PvSAMS1-F3: 5’- CTTGATATACCCCTTGCTTTCATTTG-3’
PvSAMS1-R3: 5- TTCTTTCTTTCGTTGACCATTACAT-3’
如图3所示，转基因植株中内源PvSAMS1的表达量相对于野生型对照显著性降低，说明外源转入的PvSAMS1干涉片段起到了良好的干涉效果。
实施例4：转基因植株木质素含量和糖化效率分析
木质素单体测定采用乙酰溴法。当野生型和PvSAMS1-RNAi转基因植株生长至R1（Reproduction 1）时期, 取茎节研磨，经烘干后提取木质素分析，获得不同植株的木质素的含量。如图5所示，转基因植株中木质素总量显著性低于野生型。糖化效率测定使用苯酚硫酸酯法，具体测定如下：使用纤维素酶和纤维二糖酶混合物直接酶解细胞壁残留物72 h，作为对照；使用1.5% H2SO4在121 oC条件下预处理60 min，再使用相同量的纤维素酶和纤维素二糖酶混合物酶解细胞壁残留物72 h，作为处理组。酶解产物使用苯酚硫酸酯法测定可发酵糖含量（Dubois et al，Colorimetric method for determination of sugars andrelated substances. 1956, Analytical Chemistry, 28: 350-356）。糖化效率是酶解前后可发酵糖含量的差值与酶解前可发酵糖含量的比值（图6）。
序列表
<110> 中国科学院青岛生物能源与过程研究所
<120> 柳枝稷S-腺苷甲硫氨酸合成酶基因SAMS1调控木质素合成的应用
<141> 2017-12-27
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1191
<212> DNA
<213> 柳枝稷(Panicum virgatum L.)
<400> 1
atggccgcag ttgacacctt cctctttacc tcggagtctg tgaatgaggg acaccctgac 60
aagctctgcg accaggtctc agatgccgtt cttgatgctt gccttgctga ggacccagac 120
agcaaggttg cttgcgagac ctgcaccaag actaacatgg tcatggtttt tggtgagatc 180
accaccaagg ccaacgttga ctacgagaag attgttaggg agacctgccg caacatcggt 240
ttcgtgtctg cagatgttgg gcttgatgct gacaactgca aagtgcttgt gaacattgag 300
cagcagtccc ctgatattgc tcagggtgtg catggccact tcacaaagcg gcctgaggag 360
attggagctg gtgaccaggg acacatgttc gggtatgcaa ctgatgagac cccagagctg 420
atgcccctca gtcatgtcct tgccaccaag cttggtgctc gtctcactga ggtgcgcaag 480
aacgggacct gcccctggct caggcctgat gggaagaccc aggtgactgt tgagtaccgc 540
aatgagggtg gtgccatggt ccccatccgc gtccacactg tcctcatctc tacccagcat 600
gacgagacag tcaccaacga cgagattgct gctgacctca aggagcatgt catcaagccc 660
gtcatccctg agcagtacct tgatgagaag accatcttcc acctgaaccc atctggtcgc 720
tttgtcattg gtggacctca tggtgatgct ggtctcactg gccggaagat catcattgac 780
acctatggtg gctggggagc ccatggtggt ggcgctttct ctggcaagga cccaaccaaa 840
gtcgaccgaa gtggagccta cgttgcaagg caggctgcca agagcatcgt tgctaacggc 900
cttgctcgcc gcgccatcgt ccaggtctcg tatgccattg gtgtgcctga gccactctct 960
gtgtttgtcg acacatacgg tactggcacg atcccagaca aggagattct caagattgtg 1020
aaggagaact ttgacttcag gcctggcatg atcatcatca accttgacct caagaaaggc 1080
ggcaacgggc gctacctcaa gacagcagcc tatggtcact tcggaaggga cgactctgac 1140
ttcacctggg aggtggtgaa gcccctcaag tgggagaagc cttctgctta a 1191
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<213> 柳枝稷(Panicum virgatum L.)
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Gly His Pro Asp Lys Leu Cys Asp Gln Val Ser Asp Ala Val Leu Asp
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Ala Cys Leu Ala Glu Asp Pro Asp Ser Lys Val Ala Cys Glu Thr Cys
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Thr Lys Thr Asn Met Val Met Val Phe Gly Glu Ile Thr Thr Lys Ala
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Asn Val Asp Tyr Glu Lys Ile Val Arg Glu Thr Cys Arg Asn Ile Gly
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Phe Val Ser Ala Asp Val Gly Leu Asp Ala Asp Asn Cys Lys Val Leu
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Val Asn Ile Glu Gln Gln Ser Pro Asp Ile Ala Gln Gly Val His Gly
100 105 110
His Phe Thr Lys Arg Pro Glu Glu Ile Gly Ala Gly Asp Gln Gly His
115 120 125
Met Phe Gly Tyr Ala Thr Asp Glu Thr Pro Glu Leu Met Pro Leu Ser
130 135 140
His Val Leu Ala Thr Lys Leu Gly Ala Arg Leu Thr Glu Val Arg Lys
145 150 155 160
Asn Gly Thr Cys Pro Trp Leu Arg Pro Asp Gly Lys Thr Gln Val Thr
165 170 175
Val Glu Tyr Arg Asn Glu Gly Gly Ala Met Val Pro Ile Arg Val His
180 185 190
Thr Val Leu Ile Ser Thr Gln His Asp Glu Thr Val Thr Asn Asp Glu
195 200 205
Ile Ala Ala Asp Leu Lys Glu His Val Ile Lys Pro Val Ile Pro Glu
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Gln Tyr Leu Asp Glu Lys Thr Ile Phe His Leu Asn Pro Ser Gly Arg
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Phe Val Ile Gly Gly Pro His Gly Asp Ala Gly Leu Thr Gly Arg Lys
245 250 255
Ile Ile Ile Asp Thr Tyr Gly Gly Trp Gly Ala His Gly Gly Gly Ala
260 265 270
Phe Ser Gly Lys Asp Pro Thr Lys Val Asp Arg Ser Gly Ala Tyr Val
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Ala Arg Gln Ala Ala Lys Ser Ile Val Ala Asn Gly Leu Ala Arg Arg
290 295 300
Ala Ile Val Gln Val Ser Tyr Ala Ile Gly Val Pro Glu Pro Leu Ser
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Val Phe Val Asp Thr Tyr Gly Thr Gly Thr Ile Pro Asp Lys Glu Ile
325 330 335
Leu Lys Ile Val Lys Glu Asn Phe Asp Phe Arg Pro Gly Met Ile Ile
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Ile Asn Leu Asp Leu Lys Lys Gly Gly Asn Gly Arg Tyr Leu Lys Thr
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Ala Ala Tyr Gly His Phe Gly Arg Asp Asp Ser Asp Phe Thr Trp Glu
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<210> 3
<211> 456
<212> DNA
<213> 柳枝稷(Panicum virgatum L.)
<400> 3
tggacctcat ggtgatgctg gtctcactgg ccggaagatc atcattgaca cctatggtgg 60
ctggggagcc catggtggtg gcgctttctc tggcaaggac ccaaccaaag tcgaccgaag 120
tggagcctac gttgcaaggc aggctgccaa gagcatcgtt gctaacggcc ttgctcgccg 180
cgccatcgtc caggtctcgt atgccattgg tgtgcctgag ccactctctg tgtttgtcga 240
cacatacggt actggcacga tcccagacaa ggagattctc aagattgtga aggagaactt 300
tgacttcagg cctggcatga tcatcatcaa ccttgacctc aagaaaggcg gcaacgggcg 360
ctacctcaag acagcagcct atggtcactt cggaagggac gactctgact tcacctggga 420
ggtggtgaag cccctcaagt gggagaagcc ttctgc 456

Claims (6)

1.柳枝稷SAMS1基因的开放阅读框（coding sequences，CDS）序列，其特征在于：SEQ IDNO.1所示的核苷酸序列。
2.根据权利要求1所述的PvSAMS1编码的氨基酸序列，其特征在于：SEQ ID NO.2所示的氨基酸序列。
3.根据权利要求1所述的PvSAMS1的核苷酸序列中选取的一段保守区作为后续干涉载体pANIC8B-PvSAMS1-RNAi的靶序列，其序列如SEQ ID NO.3所示。
4.含有权利要求3所述的PvSAMS1-RNAi片段的植物表达载体。
5.根据权利要求4所述的植物表达载体在调控表达水平、木质素总量和糖化效率方面的应用。
6.根据权利要求4所述的植物表达载体在柳枝稷遗传改良和分子育种方面的应用。
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