本文關(guān)鍵詞： 馬鈴薯 低溫糖化 淀粉降解 α-淀粉酶 β-淀粉酶 亞細(xì)胞定位 塊莖　出處：《華中農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：馬鈴薯(Solanum tuberosum L.)是世界上最重要的非谷物糧食作物,在保證人類糧食安全方面具有重要作用。薯片和薯?xiàng)l加工產(chǎn)品在加工業(yè)中占據(jù)著主要地位,為保證原材料的持續(xù)供應(yīng),馬鈴薯塊莖經(jīng)常貯藏于低溫條件下(不高于10°C),然而低溫貯藏會(huì)導(dǎo)致還原糖積累,即為低溫糖化現(xiàn)象,使其在油炸加工過(guò)程中產(chǎn)生褐化反應(yīng),影響產(chǎn)品品質(zhì)。前人研究表明淀粉降解是低溫糖化的主要路徑之一,但是并未鑒定到對(duì)低溫糖化起特異作用的淀粉降解酶基因,淀粉降解途徑的低溫糖化機(jī)理尚不清楚。因此,本研究從全基因組角度對(duì)馬鈴薯α-淀粉酶和β-淀粉酶家族成員進(jìn)行了鑒定、功能驗(yàn)證及作用機(jī)制解析,主要研究結(jié)果如下:1.利用馬鈴薯基因組數(shù)據(jù)庫(kù),鑒定到2個(gè)α-淀粉酶、7個(gè)β-淀粉酶家族成員,它們不對(duì)稱地分布在馬鈴薯染色體上。通過(guò)不同物種的α-淀粉酶和β-淀粉酶系統(tǒng)進(jìn)化樹(shù)分析,發(fā)現(xiàn)淀粉酶進(jìn)化比較保守,α-淀粉酶被聚為3個(gè)亞家族,β-淀粉酶被聚為4個(gè)亞家族。馬鈴薯β-淀粉酶基因家族成員基因結(jié)構(gòu)分析表明,聚在相同亞家族的成員有相似的基因結(jié)構(gòu),但葡糖基水解酶結(jié)構(gòu)域的比對(duì)結(jié)果顯示,其中一個(gè)β-淀粉酶家族成員StBAM9可能不具活性。2.利用具有不同低溫糖化抗性的馬鈴薯基因型,在不同植物組織及不同貯藏溫度下的塊莖中進(jìn)行淀粉酶家族成員的表達(dá)分析,篩選到3個(gè)受低溫響應(yīng)并且在塊莖中誘導(dǎo)表達(dá)的淀粉酶,分別是α-淀粉酶StAmy23、β-淀粉酶StBAM1和StBAM9。3.為了明確StAmy23、StBAM1和St BAM9在細(xì)胞中的作用部位,分別構(gòu)建了這三個(gè)基因融合綠色熒光蛋白的表達(dá)載體StAmy23-GFP、StBAM1-GFP、StBAM9-GFP和淀粉粒標(biāo)記基因融合紅色熒光蛋白的表達(dá)載體StGBSS-RFP,通過(guò)兩種方法(基因槍法和農(nóng)桿菌介導(dǎo)的煙草瞬時(shí)表達(dá)方法),將目標(biāo)基因與標(biāo)記基因進(jìn)行共表達(dá),并與細(xì)胞質(zhì)標(biāo)記基因RFP和葉綠素自發(fā)熒光疊加分析,首次證明StAmy23定位于細(xì)胞質(zhì),StBAM1定位于質(zhì)體基質(zhì),St BAM9定位于淀粉粒。此外,又對(duì)StBAM1、StBAM9和StGBSS的葉綠體轉(zhuǎn)運(yùn)肽和截去轉(zhuǎn)運(yùn)肽的StBAM1、StBAM9和StGBSS進(jìn)行了亞細(xì)胞定位,結(jié)果表明,它們的葉綠體轉(zhuǎn)運(yùn)肽定位于淀粉粒,但是StBAM1葉綠體轉(zhuǎn)運(yùn)肽不穩(wěn)定,可能后期轉(zhuǎn)移至基質(zhì),與其全長(zhǎng)定位于質(zhì)體基質(zhì)相關(guān)。這些結(jié)果說(shuō)明StBAM1和StBAM9的定位取決于自身的葉綠體轉(zhuǎn)運(yùn)肽。4.為了研究StAmy23、StBAM1和StBAM9在馬鈴薯塊莖低溫糖化中的功能,分別構(gòu)建了StBAM1和StBAM9基因的RNA干涉表達(dá)載體及StBAM1和StBAM9雙干涉表達(dá)載體,分別遺傳轉(zhuǎn)化不抗低溫糖化的馬鈴薯品種鄂馬鈴薯3號(hào)(E3),RNAiStAmy23轉(zhuǎn)基因株系由前人獲得。分別測(cè)定了轉(zhuǎn)基因株系的葉片淀粉含量及低溫貯藏塊莖中的糖含量、薯片油炸色澤、淀粉酶活性、淀粉含量等,結(jié)果表明,StBAM1和StBAM9參與了白天的葉片淀粉降解,但是在夜晚只有StBAM9與淀粉降解有關(guān),而StAmy23在葉片淀粉降解過(guò)程中無(wú)顯著功能。在低溫貯藏的塊莖中,與對(duì)照相比,干涉St BAM1的轉(zhuǎn)基因塊莖和同時(shí)干涉StBAM1和St BAM9的轉(zhuǎn)基因塊莖中β-淀粉酶活性降低,但是干涉StBAM9的轉(zhuǎn)基因塊莖中β-淀粉酶活性沒(méi)有顯著變化。干涉StBAM1和St BAM9均可抑制淀粉降解和還原糖積累,有效改善油炸加工品質(zhì),而雙干涉轉(zhuǎn)基因的效果則更明顯,說(shuō)明StBAM1和StBAM9可能存在功能疊加。另外,可溶性淀粉含量在干涉StBAM1的轉(zhuǎn)基因塊莖中增加,但是在干涉StBAM9的轉(zhuǎn)基因塊莖中顯著降低,表明StBAM1可能通過(guò)水解質(zhì)體基質(zhì)中的可溶性淀粉來(lái)調(diào)節(jié)低溫糖化,而StBAM9可能直接作用于淀粉粒來(lái)調(diào)節(jié)低溫糖化。此外,干涉StAmy23導(dǎo)致轉(zhuǎn)基因低溫貯藏塊莖中可溶性糖原含量顯著增加,還原糖含量降低,可能是StAmy23通過(guò)降解細(xì)胞質(zhì)中的可溶性糖原來(lái)參與低溫糖化的調(diào)節(jié)。進(jìn)一步分析不同轉(zhuǎn)基因塊莖中還原糖的含量表明,StBAM9在低溫糖化中的功能最顯著,這3個(gè)淀粉酶分別在不同的亞細(xì)胞位置水解不同的底物,在馬鈴薯低溫糖化過(guò)程中發(fā)揮著不同水平的功能,首次揭示了淀粉水解路徑在低溫糖化中的貢獻(xiàn)。5.利用酵母雙雜交系統(tǒng)分析了StAmy23、StBAM1、St BAM9分別與淀粉代謝相關(guān)蛋白磷酸酶(StLSF1和StLSF2)、葡聚糖水雙激酶(StGWD)和淀粉顆粒合酶(StGBSS)的互作關(guān)系,結(jié)果表明只有StLSF2與St BAM9互作,推測(cè)StLSF2可能參與到StBAM9介導(dǎo)的淀粉降解過(guò)程中。但原核表達(dá)蛋白StBAM1、StBAM9和StLSF2體外活性分析實(shí)驗(yàn)證明,StLSF2不影響StBAM1、StBAM9的活性,StBAM1、StBAM9也不影響StLSF2的去磷酸化作用,推測(cè)StLSF2可能不是參與StBAM9水解路徑的一個(gè)重要因子。6.利用馬鈴薯抗低溫糖化材料10908-06、CW2-1和低溫糖化敏感材料E3的塊莖,經(jīng)4°C貯藏5天后構(gòu)建了酵母雜交文庫(kù)。以StBAM9和截去葉綠體轉(zhuǎn)運(yùn)肽的StBAM9-P分別作為餌蛋白,進(jìn)行酵母文庫(kù)篩選,總共篩選到63個(gè)潛在互作蛋白,其中一個(gè)潛在互作蛋白即為StBAM1。雙分子熒光互補(bǔ)和酵母雙雜交互作驗(yàn)證的結(jié)果表明,StBAM9與StBAM1互作于淀粉粒表面,首次證明了植物淀粉酶之間存在互作。因此,我們推測(cè)StBAM9與StBAM1可能形成一個(gè)復(fù)合體,將StBAM1從質(zhì)體基質(zhì)招募到淀粉粒表面,進(jìn)行進(jìn)一步的淀粉降解。
[Abstract]:Potato (Solanum tuberosum L.) is the world's most important non grain crops, which plays an important role in human food security. Potato chips and French fries products occupy the main position in the industry, in order to ensure the continued supply of raw materials, potato tubers often stored in low temperature conditions (not higher than 10 DEG C) however, low temperature storage will lead to the accumulation of reducing sugars, namely low temperature sweetening phenomenon, the browning reaction in the frying process, influence the quality of the product. Previous studies showed that starch degradation is the main path of a low temperature saccharification, but did not play to the identification of starch degrading enzyme gene specific effect on the low temperature low temperature saccharification saccharification. The mechanism of starch degradation pathway is not clear. Therefore, the research for the identification of potato alpha amylase and beta amylase family members from a genome-wide perspective, functional verification and mechanism System analysis, the main results are as follows: 1. using the potato genome database to identify 2 alpha amylase, beta amylase family members of 7, they are distributed asymmetrically in potato chromosome. By different species of alpha amylase and amylase phylogenetic analysis found that evolutionary conservative amylase, amylase can be divided into 3 subfamilies, beta amylase were clustered into 4 subfamilies. Potato beta amylase gene family member gene structure analysis showed that poly have similar gene structure in members of the same subfamily, but the ratio of glycosyl hydrolase domain showed that one member of the family of beta amylase StBAM9 may not be activated by.2. with different potato genotypes of low temperature sweetening resistance, analysis of expression of the amylase family members in different plant tissues and different temperatures in the tubers, screening 3 by low temperature response and induced expression in tuber amylase, respectively, alpha amylase StAmy23, beta amylase StBAM1 and StBAM9.3. to determine StAmy23, StBAM1 and St BAM9 site in the cell, respectively. The expression vector StAmy23-GFP was constructed, the three fusion gene of green fluorescent protein expression vector StGBSS-RFP StBAM1-GFP. StBAM9-GFP and starch granule marker gene fusion of red fluorescent protein, by two methods (the expression of biolistic and Agrobacterium mediated method, the instantaneous tobacco) target gene and marker gene co expression, and spontaneous fluorescence overlay analysis and cytoplasmic marker gene RFP and chlorophyll, for the first time that StAmy23 was located in cytoplasm, located in StBAM1 St BAM9 is located in the plastid stroma, starch grains. In addition, the StBAM1, and the chloroplast transit peptide transporter StBAM9 and StGBSS on StBAM1, StBAM9 and StGBSS. The subcellular localization results showed that the chloroplast transit peptide located in the starch grains, but StBAM1 chloroplast transit peptide is not stable, the latter may be transferred to the substrate, and its full-length located in the plastid matrix. These results indicate that StBAM1 and StBAM9 positioning depends on the chloroplast transit peptide.4. from the body in order to study the StAmy23, StBAM1 and StBAM9 in the low temperature saccharification of potato tubers, we constructed StBAM1 and StBAM9 gene RNA expression vector and StBAM1 interference and StBAM9 interference expression vector, genetic transformation were not anti cold sweetening potato varieties e-potato 3 (E3), RNAiStAmy23 transgenic lines obtained by previous researchers. The sugar content and starch content in leaves low temperature storage tubers of transgenic lines were determined in the fried potato chips color, amylase activity, the results showed that the starch content, StBAM1, and StBAM9 in the daytime The leaf starch degradation, but at night only StBAM9 and StAmy23 and degradation of starch, starch degradation in leaves had no significant function. In the low temperature storage tubers, compared with the control, reduce the beta amylase activity in transgenic tubers and BAM1 interference St StBAM1 and St BAM9 interference transgenic tubers, but transgenic tubers interference in StBAM9 beta amylase activity did not change significantly. StBAM1 and St interference BAM9 inhibited the degradation of starch and reducing sugar accumulation, improve the quality and effect of the frying process, transgenic double interference is more obvious, indicating that StBAM1 and StBAM9 may have the function of superposition. In addition, the soluble starch content increased in transgenic tubers interference in StBAM1 however, in transgenic tubers interference StBAM9 decreased significantly, indicating that StBAM1 may through the hydrolysis of soluble starch plastid matrix to adjust the temperature of sugar However, StBAM9 may act directly on the starch grains to adjust temperature saccharification. In addition, StAmy23 interference leads to soluble glycogen content in transgenic tubers increased significantly in low temperature storage, reducing sugar content reduced, soluble sugar may be adjusted by the StAmy23 degradation in the cytoplasm in the original low temperature saccharification. Further analysis showed that the reducing sugar content of different transgenic tubers in the most significant StBAM9 in the low temperature saccharification function, the 3 amylase respectively in the subcellular location of hydrolysis of different substrates, play the function of different levels in the process of low temperature sweetening in potato starch hydrolysis, reveals the first path in the low-temperature sweetening the contribution of.5. by yeast two hybrid system of StAmy23, StBAM1 St, BAM9 were associated with the metabolism of starch protein phosphatase (StLSF1 and StLSF2), dextran water dikinase (StGWD) and starch synthase (StGBSS). The interaction between StLSF2 and St, the results show that only BAM9 interaction, suggesting that StLSF2 may participate in the degradation of starch in the process of StBAM9 mediated. But the prokaryotic expression of protein StBAM1, StBAM9 and StLSF2 in vitro analysis experiments prove that StLSF2 does not affect the activity of StBAM1, the activity of StBAM9, StBAM1, StBAM9 does not affect the dephosphorylation of StLSF2 the role, suggesting that StLSF2 may not participate in the hydrolysis of StBAM9 path is an important factor of.6. using the cold induced sweetening material 10908-06, CW2-1 and low temperature sweetening sensitive material E3 tuber by 4 ~ C after 5 days storage constructed yeast hybrid library. With StBAM9 and truncated the chloroplast transit peptide StBAM9-P as bait protein, yeast library a total of 63 screening, screening of potential interacting proteins, including a potential interacting protein is StBAM1. bimolecular fluorescence complementation and yeast two hybrid interaction test showed that StBAM9 and StBAM 1, on the surface of starch granules, it is the first time that there is interaction between plant amylase. Therefore, we speculate that StBAM9 and StBAM1 may form a complex, and StBAM1 can be recruited from plastid matrix to starch granule surface for further starch degradation.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S532

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