【摘要】：【目的】構(gòu)建柑橘HD-ZIP II轉(zhuǎn)錄因子CsHB1基因的原核表達(dá)系統(tǒng),制備多克隆抗體,并檢測抗體在‘伏令夏橙’胚性愈傷體胚誘導(dǎo)階段中的特異性,為研究CsHB1基因在柑橘體細(xì)胞胚發(fā)生過程中的功能奠定基礎(chǔ)�！痉椒ā繕�(gòu)建柑橘HD-ZIP II轉(zhuǎn)錄因子CsHB1基因的原核表達(dá)載體p GEX4T-CsHB1-N,轉(zhuǎn)化大腸桿菌誘導(dǎo)目的融合蛋白的表達(dá),并制備獲得多克隆抗體anti-CsHB1-N。通過Western blot檢測抗體在原核表達(dá)系統(tǒng)和柑橘體細(xì)胞胚誘導(dǎo)階段的特異性,并分析體細(xì)胞胚誘導(dǎo)階段CsHB1蛋白水平表達(dá)的動(dòng)態(tài)變化�！窘Y(jié)果】重組原核表達(dá)載體p GEX4T-CsHB1-N在Escherichia coli中高效表達(dá)出了分子質(zhì)量約為49 ku的GST-CsHB1-N融合蛋白,并純化獲得多克隆抗體anti-CsHB1-N;經(jīng)過Western blot分析表明,多克隆抗體可與‘伏令夏橙’愈傷體胚誘導(dǎo)階段表達(dá)的目的蛋白特異結(jié)合;蛋白表達(dá)結(jié)果分析表明,在胚性愈傷組織中CsHB1蛋白呈現(xiàn)高的表達(dá)量,隨著誘導(dǎo)培養(yǎng)的進(jìn)行,呈現(xiàn)了先下降后上升的波動(dòng)變化�！窘Y(jié)論】多克隆抗體特異性好,可與‘伏令夏橙’胚性愈傷組織中目的蛋白特異性結(jié)合,可用于CsHB1基因功能分析。
[Abstract]:[objective] to construct the prokaryotic expression system of citrus HD-ZIP II transcription factor CsHB1 gene, to prepare polyclonal antibody, and to detect the specificity of the antibody in embryogenic callus induction stage. In order to study the function of CsHB1 gene in citrus somatic embryogenesis. [methods] the prokaryotic expression vector p GEX4T-CsHB1-N, of citrus HD-ZIP II transcription factor CsHB1 gene was constructed to express the fusion protein induced by Escherichia coli. The polyclonal antibody anti-CsHB1-N. was prepared. The specificity of antibody in prokaryotic expression system and citrus somatic embryogenesis was detected by Western blot. The dynamic changes of CsHB1 protein expression during somatic embryogenesis were analyzed. [results] the recombinant prokaryotic expression vector p GEX4T-CsHB1-N efficiently expressed GST-CsHB1-N fusion protein with molecular weight of about 49 ku in Escherichia coli. The polyclonal antibody anti-CsHB1-N; was purified and purified. Western blot analysis showed that the polyclonal antibody could specifically bind to the target protein expressed in the callus embryogenic stage of 'Fulingxia orange'. The results of protein expression showed that the expression of CsHB1 protein was high in embryogenic callus. With the development of induction and culture, the expression of CsHB1 protein decreased first and then increased. [conclusion] the polyclonal antibody has good specificity. It can specifically bind to the target protein in embryogenic callus of 'Volinga orange' and can be used for functional analysis of CsHB1 gene.
【作者單位】： 武漢生物工程學(xué)院應(yīng)用生物技術(shù)研究中心;華中農(nóng)業(yè)大學(xué)園藝林學(xué)學(xué)院;
【基金】：國家自然科學(xué)基金青年基金(31201611) 湖北省自然科學(xué)基金(2016CFB390)
【分類號(hào)】：S666

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