胡杨油菜素类固醇激素合成酶基因DWF4（PeDWF4）和CPD（PeCPD）在拟南芥生长发育中的作用

发布时间：2018-04-14 22:40

  本文选题：CPD + DWF4　； 参考：《兰州大学》2016年博士论文

【摘要】：油菜素类固醇(brassinosteroids,BRs)是一类发现较晚、结构特别、活性较高、作用广谱的植物生长调节物质(激素)。DWF4(dwarf4)和CPD(constitutive photomorphogenesis and dwarfism)被认为是编码BR生物合成途径中限速反应酶的基因。近二十年来,随着研究技术的发展和BRs合成缺陷与不敏感突变体的发现,有关BRs的研究取得了很多突破性的进展。在生物合成方面,关于它的合成途径已基本清楚,调控BRs合成的基因大部分也已经被克隆,但这些研究都主要集中在拟南芥、水稻、玉米等少数几种草本植物中,在其他植物,尤其是在那些生态效益和经济效益重大的木本植物中少有研究。为了了解草、木本植物间在BR的生物合成与遗传机制方面的差异,此前本实验室以胡杨(Populus euphratica)为材料,克隆出了与拟南芥CPD(At CPD)同源的c DNA序列(命名为Pe CPD),建立了拟南芥-Pe CPD转基因系(Pe CPDTL),初步研究了该基因的功能(Wu et al,2014)。本研究同样以胡杨为材料,克隆出了与拟南芥DWF4(At DWF4)同源的c DNA序列(命名为Pe DWF4),建立了拟南芥-Pe DWF4转基因系(命名为Pe DWF4-TL),并将此Pe DWF4-TL与上述的Pe CPD-TL人工杂交获得了拟南芥-Pe DWF4-Pe CPD双转基因系(Pe CP/DW-TL)。在此基础上,在完全相同的条件下,系统分析比较了这三个转基因系之间在形态学、生理学、遗传学、解剖学、生物化学、分子生物学、蛋白质组学等方面的差异,以期了解Pe DWF4在调节植物生长发育中的作用、Pe DWF4和Pe CPD的功能差异以及Pe DWF4和Pe CPD间的相互作用。主要结果如下:1生物信息学分析发现:克隆到的Pe DWF4 c DNA全长1667bp,其中包含一个完整的长1470bp的编码框,编码一个长490个氨基酸的蛋白质。该蛋白属于CYP90B家族,与At DWF4(CYP90B1)的氨基酸序列的同源性为72.53%,与Pe CPD(CYP90A)的同源性为38.95%,与某些草本植物(雷蒙德氏棉、陆地棉)CYP90B的同源性高于其与某些树木(桃树、巨桉)CYP90B的同源性。2形态学研究发现:与野生型(WT)相比,Pe DWF4-TL的叶变细变长变卷曲,叶面积减小;Pe CPD-TL的叶与WT的相似,但叶面积增大;Pe CP/DWTL的叶与Pe DWF4-TL相似,但变得比Pe DWF4-TL的更细、更长、更卷曲。Pe DWF4-TL、Pe CPD-TL与Pe CP/DW-TL的株高都显著增加,但Pe CPD-TL增高的的幅度大于Pe DWF4-TL,Pe CP/DW-TL增高的的幅度又大于Pe CPD-TL。此外,Pe DWF4-TL的花序茎变细,Pe CPD-TL的花序茎增粗,Pe CP/DW-TL的花序茎直径介于Pe DWF4-TL与Pe CPD-TL之间(与WT相近)。3遗传学分析发现:Pe DWF4-TL花期提前,Pe CPD-TL花期滞后,Pe CP/DW-TL花期介于Pe DWF4-TL与Pe CPD-TL之间(与WT相近);Pe DWF4-TL、Pe CPD-TL与Pe CP/DW-TL的果荚都增大,但是Pe CPD-TL的大于Pe DWF4-TL的,Pe CP/DW-TL的又大于Pe CPD-TL的。Pe DWF4-TL、Pe CPD-TL与Pe CP/DW-TL中果荚数目和种子产量也都降低,但Pe DWF4-TL降低的幅度大于Pe CPD-TL降低的幅度,Pe CP/DW-TL降低的幅度又大于Pe DWF4-TL降低的幅度。此外,Pe DWF4-TL中有大量畸形和败育果荚出现,Pe CPD-TL无畸形或败育果荚出现,Pe CP/DW-TL也有大量畸形和败育果荚出现,但比例超过Pe DWF4-TL。此外,Pe DWF4-TL在抽薹期、花序茎直径、果荚发育及种子产量等方面完全不同于以前Choe等(2001)建立的拟南芥At DWF4过表达系。4生理生化研究发现:与WT相比,Pe DWF4-TL光系统II的最大光化学效率(ΦPSII)、光合量子产量(PQY)、电子传递效率(ETR)降低;Pe CPD-TL的ΦPSII与PQY都升高,ETR与WT的相近;Pe CP/DW-TL的ΦPSII和PQY与WT的相近,但ETR减小。Pe DWF4-TL中叶绿素a和b(Chl a和Chl b)含量都显著降低,但Pe CPD-TL中Chl b含量升高、Chl a未变,Pe CP/DW-TL中Chl a与Chl b含量也显著降低,降低程度与Pe DWF4-TL相似。Pe DWF4-TL茎木质素和纤维素的含量均显著降低,但Pe CPD-TL的均显著升高,Pe CP/DW-TL的介于Pe DWF4-TL与Pe CPD-TL之间(与WT相似)。5显微结构观察发现:与WT相比,Pe DWF4-TL花序茎横切面面积和髓部面积减小;Pe CPD-TL花序茎横切面、木质部、韧皮部及髓部面积都明显增大;Pe CP/DW-TL茎横切面面积与WT相近,但木质部与韧皮部面积增大,髓部面积减小。6 RT-PCR和RT-q PCR检测Pe DWF4、Pe CPD、At DWF4、At CPD、At BR6OX2、At FLC、At TCP1、At GA5的转录水平发现:Pe CPD或Pe DWF4在Pe CPD-TL或Pe DWF4-TL中表达,Pe CPD与Pe DWF4在Pe CP/DW-TL中都表达,在WT中都不表达。At DWF4、At CPD、At BR6OX2、At FLC、At TCP1、At GA5在WT和所有转基因系中都表达。Pe DWF4-TL中,Pe DWF4的表达使At DWF4、At CPD、At BR6OX2、At TCP1、At FLC及At GA5的转录水平下调;Pe CPD-TL中,Pe CPD的表达使At DWF4、At BR6OX2、At TCP1及At FLC的转录水平下调,At CPD和At GA5转录水平上调。Pe CP/DW-TL中,Pe DWF4和Pe CPD的转录水平显著低于它们在Pe DWF4-TL和Pe CPD-TL中的转录水平,At DWF4、At BR6OX2、At FLC以及At TCP1的转录水平与它们在Pe DWF4-TL中的转录水平接近,但低于它们在Pe CPD-TL中的转录水平,At CPD和At GA5的转录水平介于Pe DWF4-TL与Pe CPD-TL之间。7 ELISA检测发现:与WT内源BRs含量(1.937μg g-1FW)相比,Pe DWF4-TL中内源BRs含量(2.567μg g-1FW)显著升高,Pe CPD-TL中内源BRs含量相反的显著降低(1.323μg g-1FW),Pe CP/DW-TL中内源BRs含量(2.1731μg g-1FW)低于Pe DWF4-TL中,但高于Pe CPD-TL中。8双向电泳-MALDI-TOF-TOF串联质谱分析发现:WT、Pe DWF4-TL、Pe CPD-TL与Pe CP/DW-TL中蛋白点总数分别为704、633、732和723。Pe DWF4-TL和Pe CPD-TL与WT的匹配率分别为65.56%与62.6%;Pe CP/DWTL与Pe DWF4-TL和Pe CPD-TL的匹配率分别为39.56%与41.63%。在成功鉴定的10个差异蛋白中,其中包括黑芥子酶1(N16)、类1,5-二磷酸核酮糖羧化酶(N18)、类甘氨酸羟甲基转移酶(N19)、硫代葡萄糖苷酶(R2)、内膜相关蛋白(R3)、类果糖二磷酸醛缩酶(R4)、类核糖-5-磷酸异构酶(R5)、1,5-二磷酸核酮糖羧化酶/加氧酶大亚基(R8)、Rubisco活化酶(R12)、推定(putative)蛋白(R1),其中N16与R2与抗逆有关,R8与R12与光合作用有关,其余6个功能未知。以上研究结果表明:1)Pe DWF4和Pe CPD虽然都在BRs的生物合成中起重要作用,但在调节植物生长发育方面它们的作用并不完全相同;2)在调节植物生长发育方面Pe DWF4的作用与At DWF4的作用也可能存在差异;3)在不同的生长发育阶段或组织器官中,Pe DWF4和Pe CPD可能存在复杂的协同或拮抗作用。关于Pe DWF4和Pe CPD作用及其互作的详细机理有待于进一步研究。
[Abstract]:Brassinosteroids (brassinosteroids, BRs) is a kind of special structure, found late, high activity, broad-spectrum effects of plant growth regulators (hormones).DWF4 (dwarf4) and CPD (constitutive photomorphogenesis and dwarfism) is considered to be BR encoding biosynthetic enzyme genes in the pathway of rate limiting reaction. In the recent twenty years, along with the study on the development of technology and the defects of BRs synthesis and insensitive mutants revealed that great advances have been made on the BRs. In the biosynthesis pathway, it has been clear about the regulation of BRs synthesis, most genes have been cloned, but these studies are mainly concentrated in Arabidopsis, rice, maize and a few herbaceous plants, in other plants, especially in the economic and ecological benefits of major woody plants with little research. In order to understand the grass and woody plants in BR The differences in biosynthesis and genetic mechanism, previously in our lab (Populus euphratica) for Populus material, cloned and Arabidopsis CPD (At CPD) C homologous DNA sequences (named Pe CPD), established CPD transgenic Arabidopsis -Pe (Pe CPDTL), to study the function of the gene (Wu et al, 2014). The study also Populus as materials, cloned and Arabidopsis DWF4 (At DWF4) C homologous DNA sequences (named Pe DWF4), established DWF4 transgenic Arabidopsis -Pe (named Pe DWF4-TL), and the Pe DWF4-TL and the Pe CPD-TL artificial hybrids were obtained the Arabidopsis -Pe DWF4-Pe CPD double transgenic lines (Pe CP/DW-TL). On this basis, in the same conditions, the system between the comparison and analysis of the three transgenic lines in morphology, physiology, genetics, anatomy, biochemistry, molecular biology, proteomics and other aspects of the difference Different, in order to understand Pe DWF4 in regulating plant growth and development in the role of Pe DWF4 and Pe CPD functional differences and the interaction of Pe DWF4 and Pe CPD. The main results are as follows: 1. Bioinformatics analysis showed that the cloned Pe DWF4 C: DNA is 1667bp, which contains a full length 1470bp the encoding frame, encoding a 490 amino acid protein. This protein belongs to the CYP90B family, and At DWF4 (CYP90B1) amino acid sequence homology was 72.53%, and Pe CPD (CYP90A) has 38.95% homology with some herbaceous plants (Raymond cotton, Gossypium hirsutum L.) CYP90B homology compared to certain trees (Prunus persica, Eucalyptus grandis) CYP90B morphology of the.2 homolog found: (WT) compared with the wild type, Pe DWF4-TL thinner and longer curly leaves, smaller leaf area; leaf WT and Pe similar to CPD-TL, but increased leaf area; leaf CP/DWTL and Pe DWF4-TL Pe similar, But more than Pe DWF4-TL thinner, longer, more.Pe DWF4-TL Pe CPD-TL curl and Pe CP/DW-TL, the plant height increased significantly, but Pe increased CPD-TL is greater than Pe DWF4-TL, Pe CP/DW-TL increased the magnitude of more than Pe CPD-TL. Pe DWF4-TL in addition, inflorescence stem thinning, Pe CPD-TL of the inflorescence stems thickening, between Pe CP/DW-TL DWF4-TL and inflorescence stem diameter between Pe Pe CPD-TL (similar to WT).3 genetic analysis showed that Pe DWF4-TL Pe CPD-TL early flowering, flowering lag between Pe CP/DW-TL DWF4-TL and Pe CPD-TL Pe between flowering (similar to WT); Pe DWF4-TL, Pe CPD-TL and Pe CP/DW-TL of the pod is but Pe increased, CPD-TL more than Pe DWF4-TL, Pe CP/DW-TL and CPD-TL.Pe than Pe DWF4-TL, the number of pods and seeds of Pe CPD-TL and Pe CP/DW-TL in production also decreased, but Pe decreased to a greater extent than DWF4-TL Pe CPD-TL reduced the magnitude of Pe CP/DW- The magnitude of TL lowering and Pe is greater than the magnitude of DWF4-TL lowering. In addition, Pe DWF4-TL has a lot of deformity and abortive pods, Pe CPD-TL malformation or abortion of pod, Pe CP/DW-TL also has a lot of deformity and abortive pods, but the proportion of more than Pe DWF4-TL. in addition, Pe DWF4-TL in the bolting stage. Inflorescence stem diameter, fruit development and seed yield were completely different from the previous Choe (2001) to establish the Arabidopsis At overexpression of DWF4.4 physiological and biochemical study found that: compared with WT, the maximum photochemical efficiency of photosystem II (Pe DWF4-TL PSII), photosynthetic quantum yield (PQY), electron transfer efficiency (ETR) PSII and PQY Pe decreased; with the increase of CPD-TL, ETR and WT Pe CP/DW-TL with similar; PSII and PQY and WT are similar, but the decrease of ETR DWF4-TL in a B and chlorophyll.Pe (Chl A and Chl b) were significantly decreased, but the Pe CPD-TL Chl B content increased. Chl a Pe CP/DW unchanged. -TL Chl A and Chl B were also significantly lower, reduce the degree of.Pe DWF4-TL stem lignin and cellulose content decreased significantly and is similar to Pe DWF4-TL, but Pe CPD-TL increased significantly, between Pe CP/DW-TL and Pe CPD-TL from Pe DWF4-TL (similar to WT).5 microstructure observation showed that compared with WT. Pe DWF4-TL inflorescence stem cross-sectional area and the pith area decreased; Pe CPD-TL inflorescence stem cross section, xylem, phloem and pith area increased; Pe CP/DW-TL stem cross-sectional area is similar to WT, but the xylem and phloem area increases, the pith area decreased DWF4 RT-PCR and.6 Pe detection, RT-q PCR Pe CPD, At DWF4, At CPD, At BR6OX2, At FLC, At TCP1, At GA5 expression: the expression of Pe CPD was found in Pe DWF4 or Pe CPD-TL or Pe DWF4-TL, Pe CPD and Pe DWF4 are expressed in Pe CP/DW-TL, in WT did not express.At DWF4, At CPD. At BR6OX2, At FLC, At TCP1, At GA5 expression of.Pe DWF4-TL in WT and all transgenic lines, the expression of Pe DWF4 At DWF4, At CPD, At BR6OX2, At TCP1, At FLC and At GA5 transcription level by Pe; CPD-TL, CPD At expression of Pe DWF4, At BR6OX2, At transcription level TCP1 At and FLC At and At CPD down regulated the transcription level of GA5 upregulation of.Pe CP/DW-TL, Pe DWF4 and Pe CPD transcription level was significantly lower than that in Pe DWF4-TL and Pe CPD-TL in At DWF4, At transcription level, BR6OX2, At and FLC transcription level of At TCP1 with their Pe in DWF4-TL transcription close, but lower than that in Pe CPD-TL in transcriptional level, At between CPD and At GA5 between Pe DWF4-TL and Pe transcription level of CPD-TL.7 and WT ELISA showed that the content of endogenous BRs (1.937 g g-1FW) compared to Pe DWF4-TL, the content of endogenous BRs (2.567 g g-1FW) increased significantly in CPD-TL, Pe the content of endogenous BRs instead 鐨勬樉钁楅檷浣，

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