CN107384954B - 一种利用OsDCL3b提高稻米中蛋白质和氨基酸含量的方法 - Google Patents
一种利用OsDCL3b提高稻米中蛋白质和氨基酸含量的方法 Download PDFInfo
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Abstract
一种利用OsDCL3b提高稻米中蛋白质和氨基酸含量的方法,该方法通过RNA干扰技术抑制水稻中OsDCL3b基因的表达,首次发现可以显著提高稻米中蛋白质、总氨基酸含量以及赖氨酸等必需氨基酸的含量,本发明可使稻米中蛋白质含量提高13.9%;总氨基酸含量提高8.6%,稻米蛋白质中第一限制性氨基酸‑赖氨酸含量提高11.9%,另外7种人体的必需氨基酸含量也有明显提高,为培育高蛋白的功能营养水稻提供一种新的分子改良途径。
Description
技术领域
本发明属于植物基因工程技术领域,涉及通过抑制水稻中OsDCL3b基因的表达以提高稻米中蛋白质和氨基酸的含量。
技术背景
水稻是世界三大粮食作物之一,全世界约有30亿人主要以稻米为食,是人类21%的能量和15%的蛋白质来源。随着世界人口的快速增长和生活质量的提升,提高稻米的产量和品质,成为人们关注的重要问题。其中,利用基因工程手段进行水稻高产和优质育种将是提高稻米产量和品质的重要手段。
蛋白质是稻米中除淀粉之外的第二大类贮藏物质,为人类提供了15%的蛋白质来源。稻米蛋白质无论是溶解性、生物价(指摄取100g蛋白质后人体所能得到的体内蛋白质的量),还是易于消化吸收方面都高于其他禾谷类作物蛋白,是一种优质植物蛋白质。但是稻米中蛋白质含量较低,是所有禾谷类作物中蛋白质含量最低的一种作物。平均蛋白质含量为9.5%,另外氨基酸是组成蛋白质的基本单元,稻米中氨基酸组成和含量决定了蛋白质的应用价值。相比其他禾本科谷物,稻米中的蛋白质的氨基酸平衡相对较好,其中赖氨酸含量相对较高,被认为是稻米蛋白质中第一限制必需氨基酸,其次为苏氨酸、蛋氨酸、色氨酸,这四种氨基酸含量高低决定稻米蛋白质品质的优劣,因此,提高稻米的蛋白质和必须氨基酸含量是水稻育种十分关注的重要课题。
稻米蛋白质属于数量性状,受到三倍体胚乳核基因、细胞质基因和二倍体母体核基因的调控,遗传过程较为复杂,目前已有许多研究通过经典遗传学方法来探讨影响稻米蛋白质含量的遗传因素,并在提高稻米蛋白质和氨基酸含量做了很多探索和尝试,如利用高蛋白含量的水稻品种通过传统杂交育种来培育高蛋白质含量的水稻品种,或利用基因工程方法将玉米或高梁的总DNA或大豆中编码蛋白基因导入水稻提高稻米蛋白质含量,通过上述方法能够提高稻米中总蛋白的含量,但有时稻米中限制性氨基酸(如赖氨酸等)提高并不明显。
植物中小ncRNA是一类大小为20-30nt的非编码RNA,主要有siRNA和miRNA。通过诱导靶基因的沉默,从而对基因的表达起调控作用,进而参与植物的生长发育和逆境适应等生物过程,而各种小ncRNA形成需要Dicer蛋白的参与,植物中的Dicer称为Dicer-like(DCL),是植物中一类重要的核糖核酸酶Ⅲ,直接的生物学功能是加工双链RNA(dsRNA),产生各种不同siRNA和miRNA等小分子非编码RNA,调控其他相关蛋白基因的表达,进而间接影响真核生物的生长发育,抗病抗虫性和逆境适应性等,水稻中已报道有OsDCL1a,OsDCL2a,OsDCL3a,OsDCL3b,OsDCL4发现都参与小RNA的生成,从而影响水稻生长发育、抗病和抗逆等性状,已有研究表明OsDCL3b表达受到抑制可以降低水稻株高,可以应用于培育矮杆水稻[储成才,等.OsDCL3b在培育矮杆水稻中的应用[P].中国专利,102559653A,2012.]。但是通过抑制水稻中OsDCL3b基因表达提高稻米中蛋白质和氨基酸含量目前尚未见报道。
发明内容
本发明的目的是提供一种利用OsDCL3b提高稻米中蛋白质和氨基酸含量的方法,利用水稻中OsDCL3b基因来提高稻米蛋白质含量和氨基酸含量。该方法通过抑制水稻中OsDCL3b基因的表达,首次发现可以显著提高稻米中蛋白质、总氨基酸含量以及赖氨酸等必需氨基酸的含量,为培育高蛋白的功能营养水稻提供一种新的分子改良途径。
本发明通过以下技术方案实现的。
本发明所述的一种利用RNA干扰技术抑制OsDCL3b表达以提高稻米中蛋白质和氨基酸含量的方法,包括以下步骤:
(1)干涉载体构建:利用引物序列(SEQ ID NO.1和SEQ ID NO.2);在水稻幼穗cDNA中扩增出OsDCL3b基因的正向干涉片段(SEQ ID NO.3),然后通过酶切与干涉载体pYL(图1)进行连接构建成中间载体pYL-1,利用引物序列(SEQ ID NO.4和SEQ ID NO.5)对pYL-1进行PCR扩增,获得反向干涉片段,通过酶切、连接将反向干涉片段与pYL-1连接后,转化TOP10F′感受态细胞,挑选阳性单菌落,摇菌后提取质粒,经引物序列(SEQ ID NO.6和SEQ ID NO.7)进行PCR扩增,扩增产物经测序验证后获得干涉载体pYL-2。
(2)将干涉载体pYL-2转化到水稻细胞或组织,获得OsDCL3b基因下调表达的高蛋白质和氨基酸含量转基因水稻。
将水稻OsDCL3b基因的干涉载体pYL-2,通过原生质体转化、Ti质粒、植物病毒载体、DNA直接转化、显微注射等常规生物技术方法转化水稻愈伤组织或者细胞,再培育成转基因水稻植株,经过荧光定量PCR或Northern blot方法检测OsDCL3b基因下调表达,获得OsDCL3b基因下调表达的水稻,经凯式定氮法测定稻米中蛋白质含量和经氨基酸自动分析仪测定大米中氨基酸含量,进而获得的高蛋白质和氨基酸含量的水稻植株。
本发明优选利用RNA干扰技术来抑制水稻OsDCL3b基因表达。
本研究首次提供一种利用水稻中OsDCL3b基因来提高稻米蛋白质含量和氨基酸含量的方法,该方法通过抑制水稻中OsDCL3b基因的表达,提高稻米中蛋白质和氨基酸的含量,为培育高蛋白质的功能营养水稻提供一种新的分子改良途径,具有重要应用前景。
本发明可以显著提高稻米中蛋白质和氨基酸的含量,可以使蛋白质含量提高13.9%;总氨基酸含量提高8.6%,稻米蛋白质中第一限制性氨基酸-赖氨酸含量提高11.9%,另外7种人体的必需氨基酸,如天冬氨酸(Asp)、苏氨酸(Thr)、缬氨酸(Val)、异亮氨酸(Ile)、亮氨酸(Leu)、苯丙氨酸(Phe)和精氨酸(Arg)的含量也有明显提高。见图4和表1。
附图说明
图1为pYL质粒结构示意图。其中,P35S:花椰菜花叶病毒(CaMV)的35S启动子;Pubi:玉米泛素蛋白基因(Ubiquitin)启动子;MCS1:多克隆位点1;MCS2:多克隆位点2;Intron:内含子;ccdB:ccdB致死基因;LacZ:LacZ基因;PLac:LacZ基因启动子;nos:nos终止子;RB:右边界;LB:左边界;kanR:卡那霉素抗性基因;HPT:潮霉素抗性基因;pBR322ori:pBR322复制起始位点;pVS1ori:pVS1复制起始位点。
图2为沉默载体pYL-2的酶切验证结果。泳道1:Marker DL15000;泳道2:BamHⅠ单酶切;泳道3:BamHⅠ和HindⅢ双酶切;泳道4:空对照;泳道5:Marker DL2000。
图3为不同OsDCL3b突变体株系和野生型(ZH11)幼穗OsDCL3b基因的表达。*表示差异显著(p<0.05);**表示差异极显著(p<0.01)。
图4为2015和2016年两年种植OsDCL3b下调表达水稻(6-1)和野生型(ZH11)稻米中蛋白质含量的比较。相同的字母代表差异不显著(p>0.05);不同的字母代表差异显著(p<0.05)。
具体实施方式
下列实施例中所用的方法如无特别说明均为常规分子生物学方法,所用引物和DNA序列为上海生物工程技术有限公司合成和制备。
实施例1:RNA干涉载体的构建和遗传转化。
(1)正向干涉片段获得:提取粳稻中花11幼穗总RNA,然后反转录成cDNA,根据OsDCL3b基因序列,利用设计引物对一:SEQ ID NO.1:5’-AATTGGATCCGGTAAAGGGAAATTGTTCC-3’(下划线序列为BamHI酶切位点)和SEQ ID NO.2:5’-CCGGAAGCTTTGCAGTTGTGTCTAAGATCA-3’(下划线序列为Hind III酶切位点)。采用RT-PCR(Reverse transcription PCR)扩增获得正向干涉片段(SEQ ID NO.3),经测序验证后用于干涉载体构建。
(2)RNA干涉载体构建:正向干涉片段(SEQ ID NO.3)和pYL载体(见图1)均经过BamH I和Hind III两种酶进行双酶切、纯化、回收后,再用T4DNA酶进行连接反应,连接产物转化大肠杆菌Top10F′感受态细胞,挑选阳性单菌落,经摇菌后提取质粒获得中间干涉载体pYL-1,采用引物对二:SEQ ID NO.4:5'-CACCCTGACGCGTGGTGTTACTTCTGAAGAGG-3'和SEQ IDNO.5:5'-ACTAGAACTGCAGCCTCAGATCT ACCATGG TCG-3'对pYL-1进行PCR扩增,获得反向干涉片段,随后再用Mlu I和Pst I酶对反向干涉片段和pYL-1进行双酶切,经纯化回收后,再用T4DNA连接酶进行连接后,转化Top10F′感受态细胞,挑选阳性单菌落,摇菌提取质粒获得干涉载体pYL-2,对pYL-2载体分别用BamH I进行单酶切和BamH I和Hind III进行双酶切验证成功后(见图2);再用引物对三:SEQ ID NO.6:5’-ACATGTTGATGTGGGTTTTACTGA-3’和SEQ IDNO.7:5’-CACTGGATCAATGTCGTGAAAG-3’进行PCR扩增,扩增产物进行测序鉴定,测定结果与预期结果一致说明干涉载体构建成功。随后将构建成功的RNAi干涉载体pYL-2转化到根癌农杆菌EHA105中,通过农杆菌介导法转化水稻愈伤组织。
引物对一:
上游引物(SEQ ID NO.1):5’-AATTGGATCCGGTAAAGGGAAATTGTTCC-3’
下游引物(SEQ ID NO.2):5’-CCGGAAGCTTTGCAGTTGTGTCTAAGATCA-3’
正向干涉片段序列(SEQ ID NO.3):
AATTGGATCCGGTAAAGGGAAATTGTTCCAAGAATTCTTTTTCAATGGAATCTTTGGTAGATTATTTCATGGTTCTCGAAAAAGTGGAGCACAAAGGGATTTTATTTTCAAAAAGGGTCATGAAATACAGTGGAACACGGAAAGCATGTACTTGCTTTTACCTTTGAGGGATTCTTCATATATCCAGGATGACCTAAGCATACACTGGGAAGCAATTGAATCTTGTGCTGGTGCAGTTGAGCAGTTGTGGAGTTCGTATCAAGGAGATGAAAATGTCATTCCTGTAAATTGTATTCCACAAAAAAGAAGAGGGGGCCAAGAAGAAATTATTCATCTGGCCAATAAGTCTCTTCATTGTTCCAGCATCAAAGATTCAGTCGTGCTATCACTGCATACAGGAAGGATATACACTGTTCTTGATTTGATCTTAGACACAACTGCAAAGCTTCCGG
引物对二:
上游引物(SEQ ID NO.4):5'-CACCCTGACGCGTGGTGTTACTTCTGAAGAGG-3'
下游引物(SEQ ID NO.5):5'-ACTAGAACTGCAGCCTCAGATCTACCATGG TCG-3'
引物对三:
上游引物(SEQ ID NO.6):5’-ACATGTTGATGTGGGTTTTACTGA-3’
下游引物(SEQ ID NO.7):5’-CACTGGATCAATGTCGTGAAAG-3’
上述农杆菌介导的遗传转化方法包括以下如下步骤:
a.愈伤组织诱导和继代。
将水稻中花11种子剥去颖壳后,放入灭菌100mL三角瓶中,先用30mL75%的酒精消毒1min,然后用0.1%升汞消毒6min,无菌水冲洗5次,灭菌处理的种子接于愈伤组织诱导培养基中,放入人工气候室中在26℃下暗培养一个月左右进行愈伤组织诱导。将从胚长出的淡黄色愈伤组织接种到继代培养基中进行扩大培养,培养条件为26℃,暗培养15-20天。
愈伤组织诱导培养基:N6大量元素和微量元素、铁盐及维生素;3.0mg/L 2,4-D;0.3g/L L-proline;0.6g/L水解酪蛋白;3%的蔗糖;0.3%phytagel;pH 5.9。
愈伤组织继代培养基:N6大量元素和微量元素、铁盐及维生素;2.0mg/L 2,4-D;0.5g/L L-proline;0.6g/L水解酪蛋白;3%的蔗糖;0.8%琼脂;pH 5.9。
b.预培养。
将继代培养一次后大小为1-3mm的愈伤组织(淡黄色,致密,颗粒状)接种预培养基中,26℃暗培养4天。
预培养培养基:1/2N6大量元素和微量元素、铁盐及维生素;3.0mg/L 2,4-D;1.2g/L L-proline;0.6g/L水解酪蛋白;2%的蔗糖;0.8%琼脂;pH 5.6,另外,灭菌后按照每升培养基加入20mL 50%的葡萄糖(预先115度灭菌30min)和乙酰丁酰酮AS(100mmol/L)1mL。
c.遗传转化。
将携带OsDCL3b基因干涉载体pYL-2的农杆菌EHA105接种到YEB液体培养基中(添加100mg/L卡那霉素霉素和25mg/L利福平),28℃下摇床振荡培养72h后,离心收集菌种,用含有100μmol/L乙酰丁香酮(AS)的农杆菌悬浮培养基重新悬浮农杆菌,使菌液的OD600为0.1,并用上述浓度的农杆菌菌液浸染经过预培养的愈伤组织,浸染时间30min,随后愈伤组织放在灭菌滤纸上吸去多余菌液,接入到共培养基(含有100μmol/L的AS)中,在19℃暗环境下共培养3天,随后用无菌水清洗愈伤组织5次,最后用含有400mg/L的头孢霉素和250mg/L羧苄青霉素的无菌水浸泡愈伤10min,然后将愈伤放置于灭菌滤纸上晾干,再接种到筛选培养基中进行培养,在26℃暗环境下培养一个月后,新长出的愈伤接种到分化培养基中进行分化,将分化出幼苗接种到生根培养基中进行培养,幼苗株高长至10cm左右时移栽到大田进行种植。
YEB液体培养基:10.0g/L胰蛋白胨;1.0g/L酵母提取物;5.0g/L蔗糖,1.03g/LMgSO4·7H2O;8.0g/L琼脂(固体培养基)pH7.0-7.4
农杆菌悬浮培养基:1/2N6大量元素和微量元素,N6铁盐和复合维生素,3.0mg/L2,4-D;0.6g/L水解酪蛋白;20g/L蔗糖;10g/L葡萄糖;100μmol/L乙酰丁酰酮(AS),pH5.2。
共培养基:1/2N6大量元素和微量元素、铁盐及复合维生素;3.0mg/L 2,4-D;0.8g/L水解酪蛋白;2%的蔗糖;0.8%琼脂;pH 5.6,另外,灭菌后冷却至50℃左右,按照每升培养基加入20mL 50%的葡萄糖(预先115度灭菌30min)和乙酰丁酰酮AS(100mmol/L)1mL。
筛选培养基:N6大量元素和微量元素、铁盐及维生素;2.0mg/L 2,4-D;0.6g/L水解酪蛋白;3%的蔗糖;0.8%琼脂;pH 5.9,灭菌后冷却至50℃左右,每升培养基加潮霉素(50mg/mL);头孢噻肟钠(250mg/mL)和羧苄青霉素(250mg/mL)各1mL。
分化培养基:MS大量元素和微量元素;MS铁盐和复合维生素;3.5mg/L 6-BA;1mg/LKT;0.4mg/L NAA;0.60g/L水解酪蛋白;15.0g/L山梨醇;30.0g/L蔗糖;3.0g/L Phytagel,pH6.0。灭菌后冷却至50℃左右,每升培养基加潮霉素(50mg/mL);头孢噻肟钠(250mg/mL)和羧苄青霉素(250mg/mL)各1mL。
生根培养基:1/2MS大量元素和微量元素;MS铁盐和复合维生素;20g/L蔗糖;3.0g/L Phytagel;pH5.9,灭菌后冷却至50℃左右,每升培养基加潮霉素(50mg/mL)1mL。
实施例2转基因阳性水稻植株检测和OsDCL3b基因下调表达植株获得。
采用CTAB法提取转基因水稻叶片的DNA后,采用潮霉素磷酸转移酶基因特异性引物(SEQ ID NO.8:5’-CTGAACTCACCGCGACGTCTGTC-3’;SEQ ID NO.9:5’-TAGCGCGTCTGCTGCTCCATACA-3’)经PCR扩增为阳性的水稻,确定为T0代转基因阳性植株;采用荧光定量PCR方法,以OsDCL3b基因特异引物SEQ ID NO.10:5’-CCAGCAGTTCAGAGATTTAC-3’;SEQ ID NO.11:5’-GAAGCGTATCACACAA GAAC-3’,以水稻的OsActin作为内参基因,其引物序列为SEQ ID NO.12:5’-ACCTTCAACACCCCTGCTAT-3’和SEQ ID NO.13:5’-CACCATCACCAGAGTCC AAC-3’检测OsDCL3b基因下调表达,而获得OsDCL3b基因下调表达的水稻植株,如图3所示。
实施例3OsDCL3b基因下调表达水稻植株的稻米中蛋白质、氨基酸含量显著提高。
收获OsDCL3b下调表达水稻和野生型(非转基因水稻)成熟种子,于45℃鼓风干燥箱中干燥一周,在室温下放置至少2个月,用实验砻谷机脱掉颖壳,获得糙米,经不锈钢植物粉碎机高速粉碎,过100目筛子获得米粉,备用。按照凯式定氮法测定糙米粉中全氮含量,然后按照5.95系数换算成稻米中蛋白质的含量。先对样品蛋白质分别进行水解后,利用L-8900型氨基酸自动分析仪(HITACH,日本)进行氨基酸含量分析,全部样品均采用平行双样分析。含量为样品干重的绝对含量。发现在2015年和2016年种植的OsDCL3b下调表达水稻(6-1)的稻米中蛋白质含量分别提高15.4%和12.4%,两年平均提高了13.9%(如图4所示);发现OsDCL3b下调表达水稻(6-1)相对于野生型(ZH11),氨基酸的总含量有明显的提高,提高了8.6%,其中稻米蛋白质第一限制性必须氨基酸-赖氨酸(Lys)含量显著提高了约11.9%,另外7种人体的必需氨基酸,如天冬氨酸(Asp)、苏氨酸(Thr)、缬氨酸(Val)、异亮氨酸(Ile)、亮氨酸(Leu)、苯丙氨酸(Phe)和精氨酸(Arg)的含量也有明显提高(如表1所示)。
表1:2015和2016年种植OsDCL3b下调表达水稻(6-1)与对照(ZH11)稻米中氨基酸含量
注:表中数值为平均值±标准差,每行中相同的字母代表差异不显著(p>0.05)每行中不同的字母代表差异显著(p<0.05)。
SEQUENCE LISTING
<110> 南昌大学
<120> 一种利用OsDCL3b提高稻米中蛋白质和氨基酸含量的方法
<130> 2017
<160> 13
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Claims (1)
1.一种利用OsDCL3b干涉载体提高稻米中蛋白质和氨基酸含量的方法,其特征是包括以下步骤:
(1)干涉载体构建:利用引物序列SEQ ID NO.1和SEQ ID NO.2;在水稻幼穗cDNA中采用RT-PCR扩增出OsDCL3b基因的正向干涉片段SEQ ID NO.3,正向干涉片段SEQ ID NO.3与干涉载体pYL均经过BamH I和Hind III两种酶进行双酶切、纯化、回收后,再用T4 DNA酶进行连接反应,连接产物转化大肠杆菌Top10F′感受态细胞,挑选阳性单菌落,经摇菌后提取质粒获得中间载体pYL-1,利用引物序列SEQ ID NO.4和SEQ ID NO.5对pYL-1进行PCR扩增,获得反向干涉片段,随后再用MluI和PstI酶对反向干涉片段和pYL-1进行双酶切、经纯化回收后,再用T4 DNA连接酶进行连接后,转化Top10F′感受态细胞,挑选阳性单菌落,摇菌提取质粒获得干涉载体pYL-2;经引物序列SEQ ID NO.6和SEQ ID NO.7进行PCR扩增,扩增产物经测序验证后干涉载体构建成功;
(2)将水稻OsDCL3b基因的干涉载体pYL-2,通过原生质体转化、Ti质粒、植物病毒载体、DNA直接转化或显微注射方法转化水稻愈伤组织或者细胞,再培育成转基因水稻植株,经过荧光定量PCR或Northern blot方法检测OsDCL3b基因下调表达,获得OsDCL3b基因下调表达的水稻,经凯式定氮法测定稻米中蛋白质含量和经氨基酸自动分析仪测定大米中氨基酸含量,进而获得的高蛋白质和氨基酸含量的水稻植株;
其中:
SEQ ID NO.1:5’-AATTGGATCCGGTAAAGGGAAATTGTTCC-3’;
SEQ ID NO.2:5’-CCGGAAGCTTTGCAGTTGTGTCTAAGATCA-3’;
SEQ ID NO.4:5'-CACCCTGACGCGTGGTGTTACTTCTGAAGAGG-3';
SEQ ID NO.5:5'-ACTAGAACTGCAGCCTCAGATCT ACCATGG TCG-3';
SEQ ID NO.6:5’-ACATGTTGATGTGGGTTTTACTGA-3’;
SEQ ID NO.7:5’-CACTGGATCAATGTC GTGAAAG-3’。
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