CN113774080B - 一种利用miR156创制高花色素园艺观赏杨树的方法 - Google Patents
一种利用miR156创制高花色素园艺观赏杨树的方法 Download PDFInfo
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Abstract
本发明公开了一种利用miR156创制高花色素园艺观赏杨树的方法，属于植物基因工程技术领域。利用前体miR156b构建超表达载体，通过农杆菌介导法，将超表达载体转化杨树叶片，在杨树中过量表达miR156。所获得的转基因植株miR156表达量显著提高，花色素含量显著增加，植株矮化，叶片和分枝增多，进而提高了杨树的园艺观赏价值。
Description
技术领域
本发明涉及植物基因工程技术领域，更具体的说是涉及一种利用miR156创制高花色素园艺观赏杨树的方法。
背景技术
园艺景观树木的观赏性主要体现在树形和色彩方面。杨树高大挺拔、叶大荫浓、生长较快、适应性强，是优良的绿化树种，具有一定的观赏价值。然而，受部分绿化空间高度的限制，以杨树作为景观植物需要进行修剪以使个体植株的高度符合整体景观和生态的要求；另外，杨树的色彩也较为单一；因此，需要从树形与色彩入手寻求提高杨树的园艺观赏价值的方法。
microRNA(miRNA)是一类广泛存在于动植物中的内源性非编码小RNA，长度大约为21-24nt。通过一系列的合成和作用机制，其可以在转录后降解mRNA或者通过抑制蛋白翻译来行使生物学功能，对植物和动物的基因表达具有负调控作用。MicroRNA参与植物生长发育、非生物/生物反应、代谢物生物合成等重要的生物过程。
因此，如何利用microRNA进行杨树生长与代谢调控，提高杨树的园艺观赏价值是本领域技术人员亟待解决的问题。
发明内容
有鉴于此，本发明提供了一种利用miR156创制高花色素园艺观赏杨树的方法。
为了实现上述目的，本发明采用如下技术方案：
苜蓿miR156在调控杨树花色素合成中的应用，miR156的成熟序列如SEQ ID NO.1所示。
优选地，通过在杨树中超表达苜蓿miR156提高杨树中花色素含量。
苜蓿miR156的前体miR156b在调控杨树花色素合成中的应用，前体miR156b核苷酸序列如SEQ ID NO.2所示。
苜蓿miR156的成熟序列为前体miR156b序列自5’段6bp-25bp的一段RNA序列。
优选地，利用前体miR156b构建超表达载体，将超表达载体转入杨树中，通过在杨树中超表达苜蓿miR156提高杨树中花色素含量。
一种利用miR156创制高花色素园艺观赏杨树的方法，在杨树中超表达苜蓿miR156。
优选地，上述方法包括如下步骤：
(1)利用前体miR156b构建超表达载体；
(2)通过农杆菌介导法，将超表达载体转化杨树叶片，在杨树中过量表达miR156。
进一步地，超表达载体的具体构建方法如下：
1)以苜蓿R108的DNA做模板，用HiFi高保真扩增酶和引物pre-MtmiR156b-F、pre-MtmiR156b-R克隆苜蓿miR156的前体miR156b。
pre-MtmiR156b-F：CACCCTCCTCAGACAACAACAAG，SEQ ID NO.4；
pre-MtmiR156b-R：CTGGTGGTAGGATTTTTGTCA，SEQ ID NO.5。
2)通过XcmI酶切pCXSN质粒。
3)用T4连接酶将步骤1)中的扩增片段和步骤2)酶切的pCXSN连接，得到含有前体miR156b的超表达载体pCXSN-pre-MtmiR156b。
由上述技术方案可知，本发明在杨树中过量表达miR156，能够使花色素含量显著提高，植株的茎、叶缘、叶柄表皮均呈现红色，还可矮化植株，增加分枝，叶片小而茂密，进而提高了杨树的园艺观赏价值。此外，花青素可保护植物免受紫外线辐射影响和病原体侵入，是一种较好的抗氧化剂，可提高植物的抗逆性。同时，除花青素外，植物体内多种黄酮类物质的含量也得到提高，木质素含量减低，这对改善植物的品质，提高植物的应用价值也具有重要意义。
附图说明
图1所示为实施例1荧光定量PCR鉴定结果；
其中，
a为转基因植株(TG-1、TG-2、TG-3)与野生型植株(WT)前体miR156b表达量比较；
b为转基因植株(TG-1、TG-2、TG-3)与野生型植株(WT)成熟miR156表达量比较；
图2所示为实施例1野生型植株(WT)与miR156表达上调转基因植株(TG-1、TG-2)的株型比较；
图3所示为实施例2中转基因植株与野生型植株花青素含量比较；
其中，
a.野生型植株(WT)茎表皮颜色为绿色；
b.miR156表达上调转基因植株(TG-1)茎表皮颜色为红色；
c.野生型植株(WT)叶缘表皮颜色为绿色，miR156表达上调转基因植株(TG-1)叶缘表皮呈现红色；
d.野生型植株(WT)叶柄表皮颜色为绿色；
e.miR156表达上调转基因植株(TG-1)叶柄表皮呈现红色；
f.野生型植株(WT)和miR156表达上调转基因植株(TG-1)茎横切面比较；
图4所示为miR156表达上调转基因株系(miR156OE)与野生型植株(WT)的苯丙烷类代谢物含量对比。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅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1miR156表达上调的杨树转基因植株获得
1.pre-MtmiR156b过量表达载体构建
(1)以苜蓿R108的DNA做模板(模板部分序列如SEQ ID NO.3所示)，用HiFi高保真扩增酶和引物pre-MtmiR156b-F、pre-MtmiR156b-R克隆苜蓿miR156的前体miR156b。
pre-MtmiR156b-F：CACCCTCCTCAGACAACAACAAG，SEQ ID NO.4；
pre-MtmiR156b-R：CTGGTGGTAGGATTTTTGTCA，SEQ ID NO.5。
(2)通过XcmI酶切pCXSN质粒。
(3)用T4连接酶将步骤(1)中的扩增片段和步骤(2)酶切的pCXSN连接，得到含有前体miR156b(SEQ ID NO.2所示)的超表达载体pCXSN-pre-MtmiR156b。
2.转基因植株的获得
将超表达载体pCXSN-pre-MtmiR156b，通过农杆菌EHA105的介导，遗传转化到杂交杨树品种84K中，从而实现上调miR156的表达。转化、侵染方法如下：
(1)挑选健康的无菌苗叶片；
(2)用手术刀在滤纸上将叶片划出伤口；
(3)将50mLOD600约为0.6的含有阳性质粒载体的农杆菌EHA105离心后与叶片在40mL液体转化培养基中侵染10-15min，期间不时摇晃。
(4)将侵染后的叶片用滤纸吸干菌液，倒置铺在共培养培养基上，避光放置2天；
(5)将叶片撕开并转移到含有2mg/L潮霉素的筛选培养基上，暗培养两周，待愈伤长出后，继代到新的筛选培养基上，两周继代一次，直至长出直径约0.5cm的愈伤；
(6)转移愈伤组织至含有1.5mg/L潮霉素的分化培养基上，三周继代一次，直至长出1cm小苗；
(7)将小苗切下，转到含有1.5mg/L潮霉素的生根培养基(MS粉2.215g，蔗糖10g，调节pH至6.0，琼脂6.5g。121℃，高压灭菌15min。Tim300mg，Hyg1.5mg)中。
(8)在生根培养基长至壮苗时移栽到盆中。
其中，各培养基组成如下：
液体转化培养基：大量元素溶液1(100×)10mL，大量元素溶液2(100×)10mL，大量元素溶液3(100×)10mL，微量元素溶液1(200×)5mL，微量元素溶液2(500×)2mL，有机成分溶液(1000×)1mL，肌醇0.1g，蔗糖20g，2-吗啉乙磺酸(MES)0.5g，葡萄糖酸钙0.65g，2,4-二氯苯氧乙酸(2,4-D)1mg，激动素(KT)0.1mg，调节pH至5.6。121℃，高压灭菌15min。
共培养培养基：大量元素溶液1(100×)10mL，大量元素溶液2(100×)10mL，大量元素溶液3(100×)10mL，微量元素溶液1(200×)5mL，微量元素溶液2(500×)2mL，有机成分溶液(1000×)1mL，肌醇0.1g，蔗糖20g，MES 0.5g，葡萄糖酸钙0.65g，调节pH至6.0，琼脂7.8g。121℃，高压灭菌15min。
筛选培养基：大量元素溶液1(100×)10mL，大量元素溶液2(100×)10mL，大量元素溶液3(100×)10mL，微量元素溶液1(200×)5mL，微量元素溶液2(500×)2mL，有机成分溶液(1000×)1mL，肌醇0.1g，蔗糖20g，MES 0.5g，葡萄糖酸钙0.65g，2,4-D 1mg，KT 0.1mg，调节pH至6.0，琼脂7.8g。121℃，高压灭菌15min后添加特美汀(Tim)300mg、潮霉素B(Hyg)1.5mg。
分化培养基：大量元素溶液1(100×)10mL，大量元素溶液2(100×)10mL，大量元素溶液3(100×)10mL，微量元素溶液1(200×)5mL，微量元素溶液2(500×)2mL，有机成分溶液(1000×)1mL，肌醇0.1g，蔗糖20g，MES 0.5g，葡萄糖酸钙0.65g，6-苄氨基嘌呤(6-BA)0.5mg，1-萘乙酸(NAA)0.05mg，调节pH至6.0，琼脂7.8g。121℃，高压灭菌15min后添加Tim300mg、Hyg1.5mg。
培养基中各溶液如表1所示。
表1
3.miR156表达上调的转基因植株确定
(1)使用Trizol法提取培育10个月大的转基因植株顶端叶片和部分茎秆的RNA，反转录得到cDNA，并以野生型植株作为对照；
(2)使用pre-miR156b-qRT-F和pre-miR156b-qRT-R进行实时荧光定量PCR鉴定所得cDNA中miR156b前体的表达量；
实时荧光定量PCR 20μL反应体系：模板cDNA 2μL，pre-miR156b-qRT-F 0.5μL，pre-miR156b-qRT-R 0.5μL，2×SYBR Premix Ex TaqTM 10μL，ddH2O 7μL。
反应程序：95℃预变性30s；95℃变性5s，60℃退火、延伸30s，45个循环。溶解曲线测定：65℃至95℃。基线与循环阈值(Ct值)由Light Cycler 480软件自动生成。
pre-miR156b-qRT-F：CTTTCGTGTATGATGTTTCATTCTC，SEQ ID NO.6；
pre-miR156b-qRT-R：GCGTTCACATACCATTTTTACAA，SEQ ID NO.7；
结果如图1所示。
(3)使用miR156-qRT-F和miR156-qRT-R(成熟序列的检测引物，miRNA定量的通用引物)进行实时荧光定量PCR鉴定所得cDNA中成熟miR156的表达量；
miR156-qRT-F：CGCGCGTGACAGAAGAGAGT，SEQ ID NO.8；
miR156-qRT-R：AGTGCAGGGTCCGAGGTATT，SEQ ID NO.9；
实时荧光定量PCR 20μL反应体系：模板cDNA 2μL，miR156-qRT-F 0.5μL，miR156-qRT-R 0.5μL，2×SYBR Premix Ex TaqTM 10μL，ddH2O 7μL。
反应程序：95℃预变性30s；95℃变性5s，60℃退火、延伸30s，45个循环。溶解曲线测定：65℃至95℃。基线与循环阈值(Ct值)由Light Cycler 480软件自动生成。
如图1所示，相较于野生型植株，转基因植株的miR156、前体miR156b的表达量均出现显著上调。
进一步地，如图2所示，相较于野生型植株，miR156表达上调的转基因植株矮化了近80％，且叶片增加了5-10倍，分枝增加了约30-40倍，进而提高了杨树的园艺观赏价值。
实施例2：miR156表达上调的杨树转基因植株花青素含量测定
选择10个月大的野生型植株和过表达miR156的转基因植株，于杨树顶端取样，送迈维代谢进行代谢组学分析。
如图3所示，miR156表达上调的杨树转基因植株的茎、叶缘和叶柄的表皮呈现红色，而野生型植株均为绿色，可见，转基因植株茎、叶缘和叶柄处花青素含量均有提高。
进一步地，如图4所示，代谢组学分析结果表明，转基因植株中花青素含量相对于野生型植株显著提高。与此同时，花青素所属的苯丙烷类代谢途径中的其它黄酮类物质含量在转基因植株中也显著升高，木质素含量降低，这有利于提高植物的品质和抗逆性能。
本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
序列表
<110> 中国科学院青岛生物能源与过程研究所
<120> 一种利用miR156创制高花色素园艺观赏杨树的方法
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> RNA
<213> Artificial
<400> 1
ugacagaaga gagugagcac 20
<210> 2
<211> 95
<212> RNA
<213> Artificial
<400> 2
ggaggugaca gaagagagug agcacacaug guacuuucgu guaugauguu ucauucucga 60
agcuaugugu gcucacucuc uaucugucac cccau 95
<210> 3
<211> 3810
<212> DNA
<213> Artificial
<400> 3
ttgaaaatgg acaatgaaat ttgaaccgta tgaactcctt cccactaggg cttataagat 60
gctttggtaa caacatagag taagtgacat tgtggttcgg attgttggag tatatacgta 120
gctgataatt tacactcacc taaaaatatt aaggagtaaa ttaacaactt acacttatat 180
aaaaccataa ggagtaagtt agcaaaatat tgatcaatca ttttataaac taagtattga 240
tcattgttta gcataagcta atctctgtta agaaaatggt tggacacaca tggtgagttc 300
tatctcgagt gagttttctt tgtatacgac aaagataaag tttttatatt cttttttttc 360
taatttattt gtttttcccc ctcacattat gtaactttta cggtaaatta taattaaaat 420
ggtgttggac acacaagatg agttctatct cgagtgagtt ttctccgcat acgacaaaga 480
taaagtttct atatactttt ttttctaatt tatttgtttt ttccccgcac attatgtaac 540
ttttacagta aattatattt aaaatggcgt tggatacact aggtgagttc tatctcgagt 600
gagttttctt cgtatacgat aaagataaag tttttatata cctttttttt ccattttatt 660
tgtttttccc cttacattat gtaactttga cagtaaatta taattaaaat ggcaatggaa 720
gacatacccc acatttattc cttccatact tatacatgat gcaatcaatt attttatcct 780
tttgggctct atataaaccc ttgtatcctt cttcatctcc tcagacaaca acaagtttct 840
catttgttcc atagaaaagg aacacttagc ttctttgttg tagaatttac agagatatgg 900
tttgtttgtt attttctttt cctcctaacc ctccatcccc tatttcagaa ataatttata 960
agggttagca tagtttgatg caatacttcc aagcttaagg aagggtgatg gacgcatatc 1020
agattcaatc gagagtaagg gaggtgacag aagagagtga gcacacatgg tactttcgtg 1080
tatgatgttt cattctcgaa gctatgtgtg ctcactctct atctgtcacc ccatcaccat 1140
ctttttcttt aattcaattc aatcccttta attatattgt tttctaatac tatgcatatc 1200
tgtttatgca tcatatctca tattatgcat ctcagctaca ttttcttttc atatcatata 1260
tcattcttta tatatatggg gtacttttta ggctgatctg atcagtttca tgaaaatcat 1320
ttctgattta ggtctgccaa gttttgtaaa aatggtatgt gaacgccagc gccgttgtgg 1380
ccttaacatc catgtttttt tatgtctttg tgagcaaatt aatgcaggca ttagttgtat 1440
ttgactgtga ttttccataa tatgatgcaa ttatagcata attgcgacca caatttaaaa 1500
ttaagattaa tttatgtatg aaagacatgt gtgtgtctag atgcagaagc tattttcaca 1560
attatgaata ttgactctaa taattaatta atgaatttgg atcttatact ctcagtttaa 1620
agctttgaca aaaatcctac caccaggtga ggtgatctca tggttgagat gcaatatata 1680
gtttattctt agcatatgat gttttatatg aaaaattgta tgtaaaaaaa accatgctta 1740
atttttgtgc acctaatttt gaattcattt accaaactgt tctattaggg tttggaatct 1800
gtatgcacaa gaagcaaact gataaacaat ctggtttgtt taactctttg agaattctaa 1860
caatgagttc tatctcttcg agaaagtagt tcatttcatg cttttaattt ctattgacat 1920
acataagata aagtgcagat ctattattat tattattatt ataaatttat agtttaccta 1980
ttatatatat aaaatacatt ccttactcta ataagggagt caaactcctg atattgtaga 2040
atgaaactta atttatcttt tttgtcttat atatatgcta gaacacaata gggttctaat 2100
ttataccatg tataggtttt taatccctca caggtttcct caaatataaa tactcaagct 2160
cctttgaggt tgtgaaatgt gaagatcaat tgaaatcatc cataggctcc agcagaaagc 2220
agaagcttca atggtgtggg aaaataaaac aataatatgt actttttttc ctttaatttg 2280
aggttaacaa atgaagatgg aaattaatga ggaaattaag ggtgttgttc tggtcaattc 2340
atcttgttaa ttatattatt atgatgaaga agatgtatgc atatgtattt gtttttgaaa 2400
aaataagtga tggcatatga taataaagat ctatatattc acaaaacaaa ttcacaatag 2460
tgcattaagc ttttacaata tacatatatt aattgtttta ttcattgagg gtgtgaaagg 2520
gtaaacattt tagggaagaa tgatgttgat tgatgtaact caattttgaa tcaaatctct 2580
gtctatttta acttactacg catgtgtgaa tatgtgaaac ttcaaagtgt tgtttttaag 2640
cgattgtgaa tatgtgattc tgccaataaa aacacacaca cacacacaca tatatatata 2700
tatatatata tatatatata tatatatata tatatatata tatatatata tatatatgcg 2760
cgcgcgttaa ttagatgatg gatattaagg ctcttctaag gtttaaattt tctattcaca 2820
aggtacatgc agcttagaat ggcaatggaa gacatacccc acatttattc cttccatact 2880
tatacatgat gcaatcaatt attttatcct tttgggctct atataaaccc ttgtatcctt 2940
cttcatctcc tcagacaaca acaagtttct catttgttcc atagaaaagg aacacttagc 3000
ttctttgttg tagaatttac agagatatgg tttgtttgtt attttctttt cctcctaacc 3060
ctccatcccc tatttcagaa ataatttata agggttagca tagtttgatg caatacttcc 3120
aagcttaagg aagggtgatg gacgcatatc agattcaatc gagagtaagg gaggtgacag 3180
aagagagtga gcacacatgg tactttcgtg tatgatgttt cattctcgaa gctatgtgtg 3240
ctcactctct atctgtcacc ccatcaccat ctttttcttt aattcaattc aatcccttta 3300
attatattgt tttctaatac tatgcatatc tgtttatgca tcatatctca tattatgcat 3360
ctcagctaca ttttcttttc atatcatata tcattcttta tatatatggg gtacttttta 3420
ggctgatctg atcagtttca tgaaaatcat ttctgattta ggtctgccaa gttttgtaaa 3480
aatggtatgt gaacgccagc gccgttgtgg ccttaacatc catgtttttt tatgtctttg 3540
tgagcaaatt aatgcaggca ttagttgtat ttgactgtga ttttccataa tatgatgcaa 3600
ttatagcata attgcgacca caatttaaaa ttaagattaa tttatgtatg aaagacatgt 3660
gtgtgtctag atgcagaagc tattttcaca attatgaata ttgactctaa taattaatta 3720
atgaatttgg atcttatact ctcagtttaa agctttgaca aaaatcctac caccaggtga 3780
ggtgatctca tggttgagat gcaatatata 3810
<210> 4
<211> 23
<212> DNA
<213> Artificial
<400> 4
caccctcctc agacaacaac aag 23
<210> 5
<211> 21
<212> DNA
<213> Artificial
<400> 5
ctggtggtag gatttttgtc a 21
<210> 6
<211> 25
<212> DNA
<213> Artificial
<400> 6
ctttcgtgta tgatgtttca ttctc 25
<210> 7
<211> 23
<212> DNA
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gcgttcacat accattttta caa 23
<210> 8
<211> 20
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<213> Artificial
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cgcgcgtgac agaagagagt 20
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agtgcagggt ccgaggtatt 20
Claims (4)
1.苜蓿miR156在调控杨树花色素合成中的应用，其特征在于，
通过在杨树中超表达苜蓿miR156提高杨树中花色素含量；
miR156的成熟序列如SEQ ID NO.1所示。
2.苜蓿miR156的前体miR156b在调控杨树花色素合成中的应用，其特征在于，
利用前体miR156b构建超表达载体，将所述超表达载体转入杨树中，通过在杨树中超表达苜蓿miR156提高杨树中花色素含量；
所述前体miR156b核苷酸序列如SEQ ID NO.2所示。
3.一种利用miR156创制高花色素园艺观赏杨树的方法，其特征在于，
在杨树中超表达苜蓿miR156；
miR156的成熟序列如SEQ ID NO.1所示。
4.根据权利要求3所述的一种利用miR156创制高花色素园艺观赏杨树的方法，其特征在于，包括如下步骤：
(1)利用前体miR156b构建超表达载体；
所述前体miR156b核苷酸序列如SEQ ID NO.2所示；
(2)通过农杆菌介导法，将超表达载体转化杨树叶片，在杨树中过量表达miR156。
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