CN113736802B - 一种提高杨树木材产量的方法 - Google Patents
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Abstract
本发明专利涉及分子生物学、基因工程技术等领域，具体涉及从杨树南林895品种中鉴定出一段具有调控功能的DNA序列，对其功能和应用进行了研究。其cDNA序列如序列表1所示。本发明还公开了利用该片段促进杨树生长和调控杨树产量的方法。
Description
技术领域
本发明属于基因工程技术领域，具体涉及转化一个带有杨树PdCPD1.2(Potri.010G189800.2)基因3’UTR片段序列的基因沉默载体在提高植物产量中的应用。
背景技术
杨树是世界温带地区最主要的人工林树种之一，具有生长速度快、适应性强、成材早和木材蓄积量大等优点，而且杨树遗传背景丰富、易于快速无性化扩繁和遗传转化，随着毛果杨和胡杨全基因组测序的完成，使得杨树成为理想的木本模式植物。因而，以杨树为研究对象，研究提高杨树产量的方法对提高木材产量具有重要的意义。
油菜素内酯(BRs)属于甾醇类激素，是六大激素之一。BR能够被植物细胞膜上的受体BRI1识别，通过一系列磷酸化和去磷酸化过程将信号传递到两个同源的中心调控枢纽BZR1和BES1，在不同组织不同时期激活或抑制下游几千个靶基因精确控制细胞伸长、木质部发育、顶端优势、器官衰老和逆境胁迫等生长发育和逆境胁迫过程。当前，BR调控木质部发育的研究在拟南芥和水稻中有一些报道。鉴定拟南芥和水稻突变体发现阻断BR合成和信号转导导致植株矮小、茎秆木质部减少。而且拟南芥BZR1和BES1冗余调控木质部和韧皮部细胞分化。精细检测拟南芥茎秆发现BRs主要在形成层和正在发育木质部积累。这些结果表明BR影响草本植物木质部细胞分化。
CPD/CYP90A1是一个细胞色素单加氧化酶，催化含有3β羟基或C-22羟基和 C22、C23位双羟基的BRs合成中间产物C-3位的氧化反应，在化合物6-deoxoCT向6- deoxoTE及CT向TE转化的过程中起重要作用。CPD对不同底物的催化效率不同，其对含有Δ5-双键及含C-22羟基侧链底物的催化效率明显高于含C-22、C-23双羟基侧链的底物，加之发现DET2对含有单羟基或双羟基侧链底物具有更高的亲和度，暗示CPD介导的早期C-22羟基化途径是BRs合成的主要途径。在生物学功能方面，拟南芥CPD基因两个不同插入位点突变体cpd和cbb3都表现出BR缺失的典型特征：细胞伸长受阻，在暗培养下下胚轴变短、顶端回钩缺失。这些特征比BR合成酶DET2的突变体det2还要严重，加上CPD基因突变可阻碍DET2底物积累，说明CPD在DET2的上位。进一步研究发现 CPD基因能够被BR信号反馈调控。当BR信号被激活时，BR特异转录因子BZR1和 BES1去磷酸化，从而在转录水平直接抑制CPD表达，减少BR合成。因此通过改造CPD 基因可能会改变植物的BRs合成，进而影响植物的生长。
发明内容
本发明的目的是提供一种提高杨树产量的方法。
为实现上述目的，本发明采用技术方案为：
本发明第一方面，提供一种杨树PdCPD1.2基因的3’UTR片段，其序列如序列表1所示。
本发明第二方面，提供一种包含杨树PdCPD1.2基因3’UTR片段的基因沉默载体，所述载体包括微生物表达载体和植物表达载体。
本发明第三方面，提供一种杨树PdCPD1.2基因3’UTR片段在提高杨树产量的应用。
本发明从杨树转录组数据中发现一个CPD编码基因PdCPD1.2(Potri.010G189800.2)在发育木质部(Developing xylem)中大量表达，意味着该基因可能参与木材形成。构建RNAi载体抑制PdCPD1.2基因3’UTR能适度激活PdCPD1表达，促进杨树生长。因而本发明有助于提高林木的生物量。
附图说明
构成本发明的一部分说明图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1为沉默PdCPD1.2基因3’UTR片段后提高杨树产量。
具体实施方式
应该指出，以下详细说明都是示例性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、部件和/或它们的组合。
为推动基因工程在提高林木产量的研究和应用，本发明第一方面，提供一种杨树PdCPD1.2基因3’UTR片段，其序列如序列表1所示。
本发明优选的技术方案中，扩增所述杨树PdCPD1.2基因3’UTR片段包括以下步骤：
(1)以杨树PdCPD1.2基因3’UTR片段为模板，设计引物；
(2)提取杨树幼苗的总RNA，然后反转录成cDNA；
(3)以上述步骤(2)所得cDNA为模板，使用高保真酶进行PCR扩增，回收纯化产物，既得。
本发明第二方面，提供一种包含杨树PdCPD1.2基因3’UTR片段的基因沉默载体，所述载体包括微生物表达载体和植物表达载体。
本发明第三方面，提供一种杨树PdCPD1.2基因3’UTR片段在提高杨树产量的应用。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案，以下将结合具体的实施例详细说明本发明的技术方案。
实施例1杨树PdCPD1.2基因3’UTR片段的克隆及RNAi载体的构建
(1)在phytozome网站下载杨树PdCPD1.2基因的基因组序列，分析其特异区段，设计引物对其进行扩增；
(2)用艾德莱RNA提取试剂盒提取杨树幼苗的总RNA，然后用全式金反转录试剂盒将 RNA反转录成cDNA；
(3)以cDNA为模板，使用高保真酶进行PCR扩增，回收纯化产物，并将其构建到RNAi载体pUCC上测序，测序结果如序列表1所示。
序列表
<110> 青岛农业大学
<120> 一种提高杨树木材产量的方法
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 352
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<220>
<221> 3’UTR
<400> 1
tcaatgtgca gcgtcgagat cgtgtccaag ccatgtaaat tattaattag agcaaaagag 60
agagcatagt gggagagatg taatcctttt agtgttggat ccaataaaag ctaaaagtaa 120
caagataaaa gcagatgtta agttctggat tattgagtat tccattagta gaggttgtgg 180
gcgggtgatg atatccaccc tcctccctgt tataaccacg cccccactaa ctcacttttc 240
ttataaaccg acgaatacac ccttgaatca aacccttctc tcagtctaac tctatcgatc 300
agactattcc tctctcttcc cttctaaccc acaccaccct aaggcctaac ca 352
Claims (3)
1.一种分离的核酸，所述核酸来自杨树PdCPD1.2基因Potri .010G189800 .2，所述核酸的序列如序列表中的序列1所示。
2.含有权利要求1所述核酸的基因沉默载体。
3.权利要求2所述的基因沉默载体的应用，其特征在于，将权利要求2的载体转入杨树，转化该载体后促进杨树生长和提高木材产量。
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