本文選題：CPD + DWF4　； 參考：《蘭州大學(xué)》2016年博士論文

【摘要】：油菜素類固醇(brassinosteroids,BRs)是一類發(fā)現(xiàn)較晚、結(jié)構(gòu)特別、活性較高、作用廣譜的植物生長(zhǎng)調(diào)節(jié)物質(zhì)(激素)。DWF4(dwarf4)和CPD(constitutive photomorphogenesis and dwarfism)被認(rèn)為是編碼BR生物合成途徑中限速反應(yīng)酶的基因。近二十年來,隨著研究技術(shù)的發(fā)展和BRs合成缺陷與不敏感突變體的發(fā)現(xiàn),有關(guān)BRs的研究取得了很多突破性的進(jìn)展。在生物合成方面,關(guān)于它的合成途徑已基本清楚,調(diào)控BRs合成的基因大部分也已經(jīng)被克隆,但這些研究都主要集中在擬南芥、水稻、玉米等少數(shù)幾種草本植物中,在其他植物,尤其是在那些生態(tài)效益和經(jīng)濟(jì)效益重大的木本植物中少有研究。為了了解草、木本植物間在BR的生物合成與遺傳機(jī)制方面的差異,此前本實(shí)驗(yàn)室以胡楊(Populus euphratica)為材料,克隆出了與擬南芥CPD(At CPD)同源的c DNA序列(命名為Pe CPD),建立了擬南芥-Pe CPD轉(zhuǎn)基因系(Pe CPDTL),初步研究了該基因的功能(Wu et al,2014)。本研究同樣以胡楊為材料,克隆出了與擬南芥DWF4(At DWF4)同源的c DNA序列(命名為Pe DWF4),建立了擬南芥-Pe DWF4轉(zhuǎn)基因系(命名為Pe DWF4-TL),并將此Pe DWF4-TL與上述的Pe CPD-TL人工雜交獲得了擬南芥-Pe DWF4-Pe CPD雙轉(zhuǎn)基因系(Pe CP/DW-TL)。在此基礎(chǔ)上,在完全相同的條件下,系統(tǒng)分析比較了這三個(gè)轉(zhuǎn)基因系之間在形態(tài)學(xué)、生理學(xué)、遺傳學(xué)、解剖學(xué)、生物化學(xué)、分子生物學(xué)、蛋白質(zhì)組學(xué)等方面的差異,以期了解Pe DWF4在調(diào)節(jié)植物生長(zhǎng)發(fā)育中的作用、Pe DWF4和Pe CPD的功能差異以及Pe DWF4和Pe CPD間的相互作用。主要結(jié)果如下:1生物信息學(xué)分析發(fā)現(xiàn):克隆到的Pe DWF4 c DNA全長(zhǎng)1667bp,其中包含一個(gè)完整的長(zhǎng)1470bp的編碼框,編碼一個(gè)長(zhǎng)490個(gè)氨基酸的蛋白質(zhì)。該蛋白屬于CYP90B家族,與At DWF4(CYP90B1)的氨基酸序列的同源性為72.53%,與Pe CPD(CYP90A)的同源性為38.95%,與某些草本植物(雷蒙德氏棉、陸地棉)CYP90B的同源性高于其與某些樹木(桃樹、巨桉)CYP90B的同源性。2形態(tài)學(xué)研究發(fā)現(xiàn):與野生型(WT)相比,Pe DWF4-TL的葉變細(xì)變長(zhǎng)變卷曲,葉面積減小;Pe CPD-TL的葉與WT的相似,但葉面積增大;Pe CP/DWTL的葉與Pe DWF4-TL相似,但變得比Pe DWF4-TL的更細(xì)、更長(zhǎng)、更卷曲。Pe DWF4-TL、Pe CPD-TL與Pe CP/DW-TL的株高都顯著增加,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的花序莖變細(xì),Pe CPD-TL的花序莖增粗,Pe CP/DW-TL的花序莖直徑介于Pe DWF4-TL與Pe CPD-TL之間(與WT相近)。3遺傳學(xué)分析發(fā)現(xiàn):Pe DWF4-TL花期提前,Pe CPD-TL花期滯后,Pe CP/DW-TL花期介于Pe DWF4-TL與Pe CPD-TL之間(與WT相近);Pe DWF4-TL、Pe CPD-TL與Pe CP/DW-TL的果莢都增大,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與Pe CP/DW-TL中果莢數(shù)目和種子產(chǎn)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中有大量畸形和敗育果莢出現(xiàn),Pe CPD-TL無畸形或敗育果莢出現(xiàn),Pe CP/DW-TL也有大量畸形和敗育果莢出現(xiàn),但比例超過Pe DWF4-TL。此外,Pe DWF4-TL在抽薹期、花序莖直徑、果莢發(fā)育及種子產(chǎn)量等方面完全不同于以前Choe等(2001)建立的擬南芥At DWF4過表達(dá)系。4生理生化研究發(fā)現(xiàn):與WT相比,Pe DWF4-TL光系統(tǒng)II的最大光化學(xué)效率(ΦPSII)、光合量子產(chǎn)量(PQY)、電子傳遞效率(ETR)降低;Pe CPD-TL的ΦPSII與PQY都升高,ETR與WT的相近;Pe CP/DW-TL的ΦPSII和PQY與WT的相近,但ETR減小。Pe DWF4-TL中葉綠素a和b(Chl a和Chl b)含量都顯著降低,但Pe CPD-TL中Chl b含量升高、Chl a未變,Pe CP/DW-TL中Chl a與Chl b含量也顯著降低,降低程度與Pe DWF4-TL相似。Pe DWF4-TL莖木質(zhì)素和纖維素的含量均顯著降低,但Pe CPD-TL的均顯著升高,Pe CP/DW-TL的介于Pe DWF4-TL與Pe CPD-TL之間(與WT相似)。5顯微結(jié)構(gòu)觀察發(fā)現(xiàn):與WT相比,Pe DWF4-TL花序莖橫切面面積和髓部面積減小;Pe CPD-TL花序莖橫切面、木質(zhì)部、韌皮部及髓部面積都明顯增大;Pe CP/DW-TL莖橫切面面積與WT相近,但木質(zhì)部與韌皮部面積增大,髓部面積減小。6 RT-PCR和RT-q PCR檢測(cè)Pe DWF4、Pe CPD、At DWF4、At CPD、At BR6OX2、At FLC、At TCP1、At GA5的轉(zhuǎn)錄水平發(fā)現(xiàn):Pe CPD或Pe DWF4在Pe CPD-TL或Pe DWF4-TL中表達(dá),Pe CPD與Pe DWF4在Pe CP/DW-TL中都表達(dá),在WT中都不表達(dá)。At DWF4、At CPD、At BR6OX2、At FLC、At TCP1、At GA5在WT和所有轉(zhuǎn)基因系中都表達(dá)。Pe DWF4-TL中,Pe DWF4的表達(dá)使At DWF4、At CPD、At BR6OX2、At TCP1、At FLC及At GA5的轉(zhuǎn)錄水平下調(diào);Pe CPD-TL中,Pe CPD的表達(dá)使At DWF4、At BR6OX2、At TCP1及At FLC的轉(zhuǎn)錄水平下調(diào),At CPD和At GA5轉(zhuǎn)錄水平上調(diào)。Pe CP/DW-TL中,Pe DWF4和Pe CPD的轉(zhuǎn)錄水平顯著低于它們?cè)赑e DWF4-TL和Pe CPD-TL中的轉(zhuǎn)錄水平,At DWF4、At BR6OX2、At FLC以及At TCP1的轉(zhuǎn)錄水平與它們?cè)赑e DWF4-TL中的轉(zhuǎn)錄水平接近,但低于它們?cè)赑e CPD-TL中的轉(zhuǎn)錄水平,At CPD和At GA5的轉(zhuǎn)錄水平介于Pe DWF4-TL與Pe CPD-TL之間。7 ELISA檢測(cè)發(fā)現(xiàn):與WT內(nèi)源BRs含量(1.937μg g-1FW)相比,Pe DWF4-TL中內(nèi)源BRs含量(2.567μg g-1FW)顯著升高,Pe CPD-TL中內(nèi)源BRs含量相反的顯著降低(1.323μg g-1FW),Pe CP/DW-TL中內(nèi)源BRs含量(2.1731μg g-1FW)低于Pe DWF4-TL中,但高于Pe CPD-TL中。8雙向電泳-MALDI-TOF-TOF串聯(lián)質(zhì)譜分析發(fā)現(xiàn):WT、Pe DWF4-TL、Pe CPD-TL與Pe CP/DW-TL中蛋白點(diǎn)總數(shù)分別為704、633、732和723。Pe DWF4-TL和Pe CPD-TL與WT的匹配率分別為65.56%與62.6%;Pe CP/DWTL與Pe DWF4-TL和Pe CPD-TL的匹配率分別為39.56%與41.63%。在成功鑒定的10個(gè)差異蛋白中,其中包括黑芥子酶1(N16)、類1,5-二磷酸核酮糖羧化酶(N18)、類甘氨酸羥甲基轉(zhuǎn)移酶(N19)、硫代葡萄糖苷酶(R2)、內(nèi)膜相關(guān)蛋白(R3)、類果糖二磷酸醛縮酶(R4)、類核糖-5-磷酸異構(gòu)酶(R5)、1,5-二磷酸核酮糖羧化酶/加氧酶大亞基(R8)、Rubisco活化酶(R12)、推定(putative)蛋白(R1),其中N16與R2與抗逆有關(guān),R8與R12與光合作用有關(guān),其余6個(gè)功能未知。以上研究結(jié)果表明:1)Pe DWF4和Pe CPD雖然都在BRs的生物合成中起重要作用,但在調(diào)節(jié)植物生長(zhǎng)發(fā)育方面它們的作用并不完全相同;2)在調(diào)節(jié)植物生長(zhǎng)發(fā)育方面Pe DWF4的作用與At DWF4的作用也可能存在差異;3)在不同的生長(zhǎng)發(fā)育階段或組織器官中,Pe DWF4和Pe CPD可能存在復(fù)雜的協(xié)同或拮抗作用。關(guān)于Pe DWF4和Pe CPD作用及其互作的詳細(xì)機(jī)理有待于進(jìn)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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