CN110184293B - 一种通过提高光合效率增加植物生物量或产量的方法 - Google Patents
一种通过提高光合效率增加植物生物量或产量的方法 Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
本发明公开了一种通过提高光合效率来增加植物生物量或产量的方法，所述方法是在植物叶绿体中过表达乙醇酸脱氢酶(GDH)基因和苹果酸合酶(MS)基因实现的。本发明利用过表达GDH基因和MS基因来提高植物光合效率，进而提高生物量或产量的方法，在同时过表达MS基因的前提下，在植物叶绿体中过表达大肠杆菌来源的GDH与在植物叶绿体中过表达绿藻来源的GDH相比，生物量或者产量增加3％‑50％。
Description
(一)技术领域
本发明涉及一种提高植物产量的方法，特别涉及一种利用光合效率提高植物产量的方法。
(二)背景技术
人类数量的增加和生活水平的提高，需要消耗更多的粮食和饲料，这就要求在有限的土地上收获更多粮食。因此，培育新的高产植物品种非常重要。
植物整体的光合作用产物全部来源于酶催化CO2转化为有机碳化合物。1,5-二磷酸核酮糖羧化酶/加氧酶(RubisCO)是卡尔文循环(Calvin-Benson(CB)cycle)中的羧化酶。由于RubisCO与CO2或O2都能反应，RubisCO与O2反应产生磷酸乙醇酸，进入光呼吸循环，光呼吸导致植物中固定的碳和氮的浪费。在全球范围内，这一过程每年将大约29GT的新鲜同化碳被重新释放到大气中(Anav A，etal.Spatiotemporal patterns of terrestrial grossprimary production:a review.Rev Geophys 2015,53:785-818.)。
为了减少光呼吸造成的损失，提高植物的光合效率，目前常用的方法是通过新的光呼吸支路来回收乙醇酸中的CO2，从而达到减少光呼吸提高光合效率的目的(Peterhansel C,Blume C,Offermann S.Photorespiratory bypasses:how can theywork？[J].Journal of Experimental Botany,2013,64(3):709-715.)。
乙醇酸脱氢酶(glycolate dehydrogenase，GDH)可以将乙醇酸转换成乙醛酸。目前用于植物转基因研究和应用的乙醇酸脱氢酶主要是来源于低等植物绿藻(Chlamydomonas reinhardtii)或者大肠杆菌。绿藻中的乙醇酸脱氢酶由一个基因编码，而大杆菌中的乙醇酸脱氢酶分别由3个基因编码的D、E、F三个亚基构成。有报到通过在土豆中过表达D、E、F三个亚基的编码基因的融合基因后，植株中的DEFp融合蛋白表达量增加，葡萄糖、果糖和蔗糖等糖分成倍增加，生物量也显著增加(Nolke G,Houdelet M,Kreuzaler F,et al.The expression of a recombinant glycolate dehydrogenase polyprotein inpotato(Solanum tuberosum)plastids strongly enhances photosynthesis and tuberyield[J].Plant Biotechnology Journal,2014,12(6):734-742.)。但是大肠杆菌来源和绿藻来源的乙醇酸脱氢酶在功能和活性方面都有显著差异，所以在转基因植物中的表现也有很大的差异。
苹果酸合酶(malate synthase，MS)能催化乙酰-CoA与乙醛酸转换成苹果酸与CoA。苹果酸合酶参与了乙醛酸循环，广泛存在于不同植物体内。有研究报道在烟草中过表达绿藻的GDH和南瓜(C.maxima)的MS可以提高其光合效率和生物量(PF South,APCavanagh,HW Liu,etal.Synthetic glycolate metabolism pathways stimulate cropgrowth and productivity in the field,Science,2019:363(6422):eaat9077.)。
但是由于大肠杆菌来源的GDH与绿藻来源的GDH的功能和活性的差异，我们发现，在增加植物的生物量或者产量的效果方面，大肠杆菌来源的GDH基因比过表达绿藻来源的GDH基因更好。在同时过表达MS基因的前提下，在植物叶绿体中过表达大肠杆菌来源的GDH与在植物叶绿体中过表达绿藻来源的GDH相比，生物量或者产量增加3％-50％。因此，在植物叶绿体中过表达大肠杆菌来源的GDH与另外一种MS是通过提高光合效率来增加植物生物量或产量的最好方法。
(三)发明内容
本发明目的是提供一种减少植物光呼吸，提高植物光合效率，提高植物生物量或产量的方法，所述方法是在植物叶绿体中过表达大肠杆菌源的GDH和任何来源的MS。本发明解决的问题是通过高活性的GDH基因和MS基因配合使用来优化植物光呼吸途径，获得生物量或产量增加更多的转基因植物。
本发明采用的技术方案是：
本发明提供一种通过提高光合效率来增加植物生物量或产量的方法，所述方法是在植物叶绿体中过表达乙醇酸脱氢酶(GDH)基因和苹果酸合酶(MS)基因实现的。优选所述乙醇酸脱氢酶GDH基因来源于大肠杆菌，所述GDH由三个独立的亚基组成(核苷酸序列为：GenBank:CP029238.1中3126522bp-3128021bp，编码的氨基酸为：GenBank:QBP03082.1；GenBank:CP029238.1中3125470bp-3126522bp，编码的氨基酸为：GenBank:QBP03081.1；核苷酸序列为：GenBank:CP029238.1中3124236bp–3125459bp，编码的氨基酸为：GenBank:QBP03080.1)，更优选所述GDH基因序列如SEQ ID NO.1中125bp-4009bp所示，氨基酸序列SEQ ID NO.2中48-1333所示。
本发明所述苹果酸合酶MS基因来源于原核生物或者真核生物，例如表1所示MS基因，优选MS基因核苷酸序列如SEQ ID NO.3、SEQ ID NO.4(11bp-1852bp)或SEQ ID NO.5所示，氨基酸序列如SEQ ID NO.6、SEQ ID NO.7和SEQ ID NO.8所示。
表1：不同物种来源的苹果酸合酶(MS)基因
进一步，所述GDH基因和MS基因在叶绿体中过表达通过信号肽介导，所述介导是将叶绿体信号肽序列融合在GDH或MS蛋白的N端；所述信号肽来源于植物RuBisCO小亚基(RbcS)或磷酸葡萄糖变位酶转运肽序列，优选信号肽的氨基酸序列如SEQ ID NO.9或SEQID NO.10所示。
进一步，所述GDH基因和MS基因在叶绿体中过表达还包括启动子，所述启动子来源于真核生物或原核生物，也可以通过人工合成获得；优选启动子组成型启动子或特异性启动子，更优选所述启动子的核苷酸序列为UBI启动子(GenBank:KR297238.1中4879bp-6876bp所示)、Act1启动子(GenBank:AY452735.1中2428bp-3797bp所示)或35S启动子(GenBank:MG719235.1中848bp-1628bp所示)。
进一步，所述GDH基因和MS基因在叶绿体中过表达还包括终止子，所述终止子来源于真核生物或者原核生物，也可以通过人工合成获得，优选所述终止子核苷酸序列为SEQID NO.11或SEQ ID NO.12所示。
本发明所述过表达可以通过分子聚合或者杂交聚合的方法实现；分子聚合是指将GDH基因和MS基因的表达框构建在同一个载体的T-DNA上，通过转基因的方法将T-DNA转入的受体植物基因组中，从而使得目标植株中同时过表达GDH基因和MS基因。杂交聚合是指分别获得在叶绿体中过表达GDH和MS基因的植株，再用传统育种的方法，将分别表达GDH基因和MS基因的植株进行杂交，获得同时过表达GDH和MS基因的植株。
进一步，所述GDH基因和MS基因在叶绿体中过表达的方法为：(1)以商业化的双元载体pCambia1300为基础，通过XhoI酶切位点把载体的hptII(hygromycin resistance)基因置换成耐草铵膦的bar基因(GenBank:MG719235.1(287bp-837bp))，置换后的载体命名为pCambia1300-bar；(2)通过EcoRI和KpnI位点把启动子与GDH基因连入pCambia1300-bar载体中，获得过渡载体pCambia1300-bar-GDH；(3)再通过KpnI和HindIII位点把启动子和MS基因连入过渡载体pCambia1300-bar-GDH中，获得终载体pCambia1300-bar-GDH-MS，即T-DNA质粒。(4)最后，通过电转的方法把T-DNA质粒转入农杆菌LB4404中，通过含有15μg/ml四环素和50μg/mL的卡那霉素的YEP固体培养基筛选出阳性克隆，筛选获得光呼吸减少，光合效率提高，生物量或产量提高的植物。
本发明所述植物包括玉米、水稻和大豆。
与现有技术相比，本发明有益效果主要体现在：
本发明提供了一种利用过表达GDH基因和MS基因来提高植物光合效率，进而提高生物量或产量的方法，在同时过表达MS基因的前提下，在植物叶绿体中过表达大肠杆菌来源的GDH与在植物叶绿体中过表达绿藻来源的GDH相比，生物量或者产量增加3％-50％。
(四)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1、载体的构建
为了构建转化载体，人工合成了大肠杆菌来源的GDH基因以及对应的终止子序列，包含叶绿体信号肽、GDH编码基因和终止子，核苷酸序列如SEQ ID NO.1所示(编码蛋白氨基酸序列为SEQ ID NO.2所示)，5'端和3'端分别设置有BamHI和KpnI位点。
人工合成了水稻的MS基因，包含叶绿体信号肽、MS编码基因和终止子，核苷酸序列如SEQ ID NO.4所示，5'端和3'端分别设置有BamHI和HindIII位点。
同时，人工合成花椰菜花叶病毒(CaMV)的35S启动子序列(GenBank:MG719235.1中848bp-1628bp所示)，5'端和3'端分别设置有KpnI和BamHI位点。人工合成玉米Ubi启动子序列(GenBank:KR297238.1中4879bp-6876bp所示)，5'端和3'端分别设置有EcoRI和BamHI位点。
为了构建可以用于农杆菌方法转化植物所用的双元载体，用商业化的双元载体pCambia1300为基础，通过XhoI酶切位点把之前的hptII(hygromycin resistance)基因置换成耐草铵膦的bar基因(GenBank:MG719235.1(287bp-837bp)，置换后的载体命名为pCambia1300-bar。
通过EcoRI和KpnI位点把Ubi启动子与GDH基因连入pCambia1300-bar载体中，获得过渡载体pCambia1300-bar-GDH。再通过KpnI和HindIII位点把35S启动子和MS基因连入过渡载体pCambia1300-bar-GDH中，获得T-DNA质粒pCambia1300-bar-GDH-MS。
作为对照，人工合成了绿藻来源的CrGDH基因，包含叶绿体信号肽、CrGDH编码基因和终止子，核苷酸序列如SEQ ID NO.13所示，5'端和3'端分别设置有BamHI和KpnI位点。
通过EcoRI和KpnI位点把Ubi启动子与CrGDH基因连入pCambia1300-bar载体中，获得过渡载体pCambia1300-bar-CrGDH。再通过KpnI和HindIII位点把35S启动子和MS基因连入过渡载体pCambia1300-bar-CrGDH中，获得T-DNA质粒pCambia1300-bar-CrGDH-MS。
最后，通过电转的方法把T-DNA质粒转入农杆菌LB4404中，通过含有15μg/ml四环素和50μg/mL的卡那霉素的YEP固体培养基筛选出阳性克隆，并保菌，用于接下来的植物转化。
YEP固体培养基组成：牛肉浸膏5g/L，酵母浸膏1g/L，蛋白胨5g/L，蔗糖5g/L，MgSO4·H2O 0.5g/L，溶剂为水，pH 7.0。
实施例2、水稻转化
转基因水稻的获得方法是采用现有技术(卢雄斌，龚祖埙(1998)生命科学10：125-131；刘凡等(2003)分子植物育种1：108-115)。选取成熟饱满的“秀水-134”种子去壳，诱导产生愈伤组织作为转化材料。取实施例1中构建好的分别含有pCambia1300-bar-GDH-MS、pCambia1300-bar-CrGDH-MS和pCambia1300-bar-GDH质粒的农杆菌划板。挑单菌落接种，准备转化用农杆菌。将待转化的愈伤组织放入OD600为0.6的农杆菌菌液中(农杆菌菌液的制备：将农杆菌接种至培养基，培养至OD600为0.6；培养基组成：3g/L K2HPO4、1g/LNaH2PO4、1g/LNH4Cl、0.3g/L MgSO4·7H2O、0.15g/L KCl、0.01g/L CaCl2、0.0025g/L FeSO4·7H2O、5g/L蔗糖、20mg/L乙酰丁香酮，溶剂为水，pH＝5.8)，让农杆菌结合到愈伤组织表面，然后把愈伤组织转移到共培养培养基(MS+2mg/L 2,4-D+30g/L葡萄糖+30g/L蔗糖+3g/L琼脂(sigma 7921)+20mg/L乙酰丁香酮)中，共培养2-3天。用无菌水冲洗转化后的愈伤，转移到筛选培养基(MS+2mg/L2,4-D+30g/蔗糖+3g/L琼脂(sigma 7921)+20mg/L乙酰丁香酮+2mM草甘膦(Sigma))上，筛选培养两个月(中间继代一次)。把筛选后，生长活力良好的愈伤转移到预分化培养基(MS+0.1g/L肌醇+5mg/L ABA+1mg/L NAA+5mg/L 6-BA+20g/L山梨醇+30g/L蔗糖+2.5g/L gelrite)上培养20天左右，然后将预分化好的愈伤组织移到分化培养基上，每天14小时光照分化发芽。2-3周后，把抗性再生植株转移到生根培养基(1/2MS+0.2mg/L NAA+20g/L蔗糖+2.5g/L gelrite)上壮苗生根，最后将再生植株洗去琼脂移植于温室，选择产量高、种子大或者生物量高等能够提高水稻产量的转基因株系，培育新品种。分别获得含上述转化载体的转基因水稻植株。
实施例3.大豆转化
这里使用的获得转基因大豆的步骤来自于已有的技术(Deng et al.,1998,PlantPhysiology Communications 34:381-387；Ma et al.,2008,ScientiaAgriculturaSinica 41:661-668；Zhou et al.,2001,Journal of NortheastAgricultural University 32:313-319)。选取健康、饱满、成熟的“天隆1号”大豆，用80％乙醇消毒2分钟，再用无菌水清洗，然后放置在充满氯气(由50mlNaClO与2ml浓HCl反应生成)的干燥器中灭菌4-6个小时。灭菌后的大豆在超净工作台里被播撒到B5培养基中，25℃条件下培养5天，同时光密度在90-150μmol光子/m2·s水平。当子叶变绿并顶破种皮，无菌的豆芽就会长出。去掉了下胚轴的豆芽在长度上被切成五五开，使得两片外植体都具有子叶和上胚轴。在子叶和上胚轴的节点处切外植体大约7-8处，即可用作被侵染的目标组织。
分别取通过实施例1构建的含有载体pCambia1300-bar-GDH-MS、pCambia1300-bar-CrGDH-MS和pCambia1300-bar-GDH的单克隆农杆菌被分开培养待用。准备好的外植体浸没在农杆菌悬浮液中共培养30分钟左右。然后，将侵染的组织上多余的细胞悬浮液用吸水纸吸收干净，再转移到1/10B5共培养培养基里25℃暗培养3-5天。
共培养的植物组织用B5液体培养基清洗，以除去多余的农杆菌，然后放置到B5固体培养基中25℃下培养5天，待其发芽。诱导发生的胚芽组织转移到含有0.1M草甘膦的B5筛选培养基中，25℃光照培养4周，期间每两周更换一次培养基。筛选出来的胚芽组织再转移到B5固体培养基中，25℃培养，待其长成小苗。随后，将转基因植株苗转移到1/2B5培养基中进行生根诱导。最后，长成的小植株经清洗去除琼脂后栽种在温室中。
实施例4：转基因水稻的鉴定
通过实施例2分别获得了载体pCambia1300-bar-GDH-MS(GM)、pCambia1300-bar-CrGDH-MS(CGM)和pCambia1300-bar-GDH(G)的转基因水稻植株。上述转基因植株与非转基因对照相比生物量和产量都有所增加，并且，GM植株在生物量或产量方面的增加幅度最大。为了进一步鉴定GM转基因植株的表现变化，我们对上述转基因植株的生物量和种子产量进行了评估，结果如下表2所示。GM水稻植物的生物量或产量比CGM和G植株增加5％-50％。
表2
| GM | CGM | G | |
| 生物量比非转基因对照增加的幅度 | 25％ | 15％ | 9％ |
| 产量比非转基因对照增加的幅度 | 18％ | 12％ | 6％ |
实施例5：转基因大豆的鉴定
通过实施例3分别获得了载体pCambia1300-bar-GDH-MS(GM)、pCambia1300-bar-CrGDH-MS(CGM)和pCambia1300-bar-GDH(G)的转基因大豆植株。上述转基因植株与非转基因对照相比生物量和产量都有所增加，并且，GM植株在生物量或产量方面的增加幅度最大。为了进一步鉴定GM转基因植株的表现变化，我们对上述转基因植株的生物量和种子产量进行了评估，结果如下表3所示。GM大豆植物的生物量或产量比CGM和G植株增加5％-50％。
表3
| GM | CGM | G | |
| 生物量比非转基因对照增加的幅度 | 21％ | 12％ | 7％ |
| 产量比非转基因对照增加的幅度 | 14％ | 8％ | 5％ |
Claims (1)
1.一种通过提高光合效率增加植物生物量或产量的方法，其特征在于所述方法是在植物叶绿体中过表达大肠杆菌的乙醇酸脱氢酶GDH基因和水稻来源的苹果酸合酶MS基因实现的；所述乙醇酸脱氢酶GDH由三个独立的亚基组成， 分别是GenBank: CP029238.1中3126522 bp- 3128021 bp，GenBank: CP029238.1中3125470 bp- 3126522 bp，GenBank:CP029238.1中3124236 bp – 3125459 bp；所述苹果酸合酶MS基因核苷酸序列如NCBIAccession Number：CAD79704所示；所述GDH基因和MS基因在叶绿体中过表达通过信号肽介导，所述信号肽来源于植物RuBisCO小亚基或磷酸葡萄糖变位酶转运肽序列；所述GDH基因和MS基因在叶绿体中过表达还包括启动子，所述启动子的核苷酸序列为UBI启动子、Act1启动子或35S启动子；所述UBI启动子核苷酸序列如GenBank: KR297238.1中4879 bp- 6876bp所示；所述Act1启动子核苷酸序列如GenBank: AY452735.1中2428 bp- 3797 bp所示；所述35S启动子核苷酸序列如GenBank: MG719235.1中848 bp- 1628 bp所示；所述植物是水稻或大豆。
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