本文關(guān)鍵詞：DPC浸種促進(jìn)棉花側(cè)根發(fā)育的激素機(jī)制及結(jié)合態(tài)ABAscFv的制備　出處：《中國農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 縮節(jié)安 棉花 生長素 單鏈抗體

【摘要】：縮節(jié)安(N,N-Dimethylpiperidinium choride,DPC)是一種在棉花上廣泛使用的植物生長調(diào)節(jié)劑,其主要的生理效應(yīng)之一是促進(jìn)側(cè)根發(fā)育。植物側(cè)根發(fā)育的好壞與營養(yǎng)物質(zhì)的吸收及抵抗低溫、干旱等逆境的能力有重要的關(guān)系。本研究以DPC浸種處理棉花種子,探討DPC促進(jìn)棉花幼苗側(cè)根發(fā)育的激素機(jī)制。主要結(jié)果如下:1.DPC浸種處理后,增加了側(cè)根原基和側(cè)根的數(shù)量,處理后4-7d,分別比對照增加了 32.1%、34.9%、21.8%和10.9%。同時通過石蠟切片觀察到DPC浸種處理后側(cè)根原基的起始階段提前。2.DPC浸種處理后,IAA含量在根和子葉中表現(xiàn)出部位差異性。處理后2和3d,顯著增加了根中的IAA含量,但降低了在子葉中的IAA和IAA結(jié)合態(tài)含量。通過免疫組化定位技術(shù),發(fā)現(xiàn)DPC浸種處理后,增加了主根中IAA在中柱的積累,對表皮和皮層沒有影響。3.DPC浸種處理后IAA主要合成基因GhTAA1、GhTAR2的表達(dá)量沒有顯著變化,但子葉中IAA結(jié)合態(tài)水解酶基因GhILR1、GhIhR3.1和GhIAR3.2的表達(dá)量上調(diào),從而加速了 IAA結(jié)合態(tài)的水解,同時IAA輸出載體GhPIN1s和GhPIN2基因表達(dá)量上調(diào),但對IAA輸入載體GhAUX1基因表達(dá)量的調(diào)節(jié)沒有規(guī)律。因此推斷根中自由態(tài)IAA的增加主要來自于子葉中IAA結(jié)合態(tài)的水解并通過PIN蛋白將IAA轉(zhuǎn)運(yùn)。4.GA3浸種處理的表型與DPC處理后的表型相反,顯著降低了側(cè)根原基數(shù)量和密度。DPC浸種處理后根中GA含量降低。GA合成基因GhCPS、GhKS、Gh20oxr1和Gh20ox2總體呈下調(diào)的趨勢,GA代謝基因Gh2ox1和Gh2ox2呈上調(diào)的趨勢。5.克隆了 DPC處理后表達(dá)量上調(diào)較大的IAA結(jié)合態(tài)的水解酶GhIAR3.1基因并進(jìn)行功能驗證,將純化的重組蛋白GST-GhIAR3.1與IAA氨基酸結(jié)合態(tài)孵育,通過HPLC檢測表明GST-GhIAR3.1能夠催化IAA-Ala與IAA-Asp生成自由態(tài)IAA,其平均水解速率分別為11.6±1.9和1.86±0.25μmolIAAreleased/min/mg,該酶不能催化IAA-Ile和IAA-Phe。通過酶促動力學(xué)分析,GST-GhIAR3.1 與 IAA-Ala 的 Vmax 和Kw 計算分別為 17.2 μmol/min/mg 和 320.67 μM。6.通過載體上自帶的Thromin酶將標(biāo)簽GST和目的蛋白GhIAR3.1分離。將目的蛋白GhIAR3.1免疫新西蘭大耳白兔和Balb/c小鼠,成功制備了兔多抗和小鼠單克隆抗體并建立雙抗夾心ELISA。用所建立的雙抗夾心ELISA測定了子葉中水解酶GhIAR3.1的含量,發(fā)現(xiàn)DPC浸種處理后2d,子葉中水解酶GhIAR3.1的含量比對照增加了 17.8%。本論文還以實驗室保存的ABA結(jié)合態(tài)雜交瘤細(xì)胞為材料克隆了 ABA結(jié)合態(tài)VH、VL基因,將VH、VL用一條短肽連接為scFv,scFv與MBP標(biāo)簽融合純化分析其結(jié)合特性,通過ELISA表明能夠結(jié)合ABA及ABA結(jié)合態(tài)。ABA、ABA-ME和ABAGE四乙酰基的10%抑制率分別為19.7、8.1和4.0 μg/ml。ABA-ME和ABAGE四乙�；囊种坡史謩e為ABA之間1.7和2.4倍。將ABA結(jié)合態(tài)的scFv建立3D同源模型,并與ABA、ABA-ME和ABAGE分子對接,結(jié)果顯示與ABA、ABA-ME 和 ABAGE的結(jié)合能量分別為-37.1-69.6 和-112.0 kcal.mol-1,ABA-ME/ABA和ABAGE/ABA的能量比率分別為1.87和3.0,與ELISA得出的結(jié)果一致。
[Abstract]:DPC (N, N-Dimethylpiperidinium, Choride, DPC) is a widely used in cotton plant growth regulator, one of the main physiological effects is to promote the development of lateral roots. The absorption and low temperature resistance and good nutrition plant lateral root development, has an important bearing ability of drought. In this study, DPC soaking treatment of cotton seeds, DPC on hormone mechanism of cotton seedling lateral root development. The main results are as follows: 1.DPC treatment, increased the number of lateral roots and lateral roots, 4-7d after treatment were increased by 32.1%, 34.9%, 21.8% and 10.9%. were observed by paraffin section and DPC soaking after initiation of lateral root primordia early.2.DPC treatment, IAA content in roots and cotyledons showed part difference. After treatment of 2 and 3D, significantly increased the IAA content in roots, but decreased in the cotyledon in IAA and I AA boundforms. Through immunohistochemical localization technology, found that DPC treatment, increased IAA in taproot column accumulation did not affect.3.DPC treatment IAA synthetic gene GhTAA1 on the epidermis and cortex, the expression of GhTAR2 did not change significantly, but in the cotyledon IAA binding state of GhILR1 hydrolase gene expression. The amount of up regulation of GhIhR3.1 and GhIAR3.2, thus accelerating the hydrolysis of IAA bound, while the IAA output vector GhPIN1s and GhPIN2 gene expression, but the input to the IAA expression vector of GhAUX1 gene in the regulation of the volume without law. Therefore conclude that increasing the free state in the roots of IAA mainly from cotyledon IAA bound hydrolysis by PIN the phenotype of DPC protein and IAA translocation of.4.GA3 after soaking in contrast, decreased the content of GA in the root of lateral root primordium number and density of.DPC treatment decreased.GA synthesis gene GhCPS, GhKS, Gh20oxr1 and Gh20ox2 showed an overall downward trend, the metabolism of GA gene Gh2ox1 and Gh2ox2 was higher in.5. was cloned after DPC treatment up-regulated IAA hydrolase GhIAR3.1 gene combined with large state and functional verification, the purified recombinant protein GST-GhIAR3.1 and IAA amino acid bound incubation by HPLC detection showed that GST-GhIAR3.1 IAA-Ala and IAA-Asp can catalyze the formation of free state IAA, the average rate of hydrolysis were 11.6 + 1.9 and 1.86 + 0.25 molIAAreleased/min/mg, IAA-Ile and IAA-Phe. of the enzyme catalyzed not by enzymatic kinetic analysis, Vmax and Kw to calculate GST-GhIAR3.1 and IAA-Ala were Thromin 17.2 mol/min/mg and 320.67 M.6. by the carrier on its own the label GST and GhIAR3.1 protein. GhIAR3.1 protein separation to immunize New Zealand white rabbits and Balb/c mice, rabbit were prepared. And anti mouse monoclonal antibody and establishment of double antibody sandwich ELISA. content in cotyledon hydrolase GhIAR3.1 was determined by the double antibody sandwich ELISA, found that DPC treatment 2D content in cotyledon hydrolase GhIAR3.1 increased by 17.8%. of the laboratory preserved ABA as material to clone the ABA bound VH states hybridoma cells with VL gene, VH, VL with a short peptide connection for scFv, purification and analysis of its binding properties of scFv and MBP fusion tag, by ELISA that can be combined with ABA and ABA combined with.ABA ABA-ME and ABAGE state, four acetyl 10% inhibition rates were 19.7,8.1 and 4 g/ml.ABA-ME and ABAGE four deacetylation rate was ABA between 1.7 and 2.4 times. The ABA bound scFv 3D homology models, and with ABA, ABA-ME and ABAGE molecular docking, with that of the ABA, the binding energy ABA-ME and ABAGE respectively. -37.1-69. The energy ratios of 6 and -112.0 kcal.mol-1, ABA-ME/ABA and ABAGE/ABA are 1.87 and 3, respectively, which are in accordance with the results obtained from ELISA.

【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S562

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