黑穗病菌脅迫下甘蔗SSH文庫構(gòu)建及差異表達(dá)基因的克隆與分析
發(fā)布時間：2018-09-04 11:15
【摘要】：甘蔗黑穗病是由黑粉菌(Sporisorium scitamineum)引起的一種氣傳真菌性病害,已經(jīng)成為世界各植蔗區(qū)的主要病害之一。甘蔗黑穗病能引起蔗莖產(chǎn)量降低和蔗糖分的減少,給甘蔗產(chǎn)業(yè)造成較為嚴(yán)重的損失。雜交育種是甘蔗抗黑穗病育種最主要也是最重要的途徑,但是,由于甘蔗抗黑穗病基因大多來源于野生種,而野生種中高產(chǎn)高糖基因與一些不利基因存在緊密連鎖的關(guān)系,使得甘蔗傳統(tǒng)雜交育種在導(dǎo)入抗病基因的同時,也導(dǎo)入了一些不良的非目標(biāo)性狀基因。因此,如果能在解析甘蔗與黑穗病菌互作的分子機理的基礎(chǔ)上,挖掘抗病關(guān)鍵基因,進(jìn)而通過轉(zhuǎn)基因手段將此類基因轉(zhuǎn)導(dǎo)入目標(biāo)甘蔗品種中,達(dá)到定向改良品種抗性的目的,既可以避免不良性狀基因的導(dǎo)入,又能保持目標(biāo)甘蔗品種原有的優(yōu)良性狀和品質(zhì)特性,同時兼具抗黑穗病的特性,是解決甘蔗抗黑穗病育種瓶頸的一種有效途徑。本研究以甘蔗品種新臺糖22號(ROC22)為實驗材料,首先,使用抑制消減雜交(suppression subtractive hybridization, SSH)技術(shù)構(gòu)建cDNA文庫；其次,利用反向Northern雜交技術(shù),篩選差異表達(dá)基因；再次,通過電子克隆和RT-PCR相結(jié)合的技術(shù),克隆目的基因；最后,在對所克隆基因進(jìn)行生物信息學(xué)分析的基礎(chǔ)上,利用實時熒光定量PCR技術(shù),檢測這些基因的組織特異性表達(dá)情況以及在黑穗病菌、SA (salicylic acid)、MeJA (methyl-jasmonate)和ABA (abscisic acid)脅迫下的表達(dá)特性,以期揭示這些候選基因在甘蔗對黑穗病抗性中的作用機制,為后續(xù)利用甘蔗基因工程手段定向改良甘蔗品種對黑穗病的抗性,提供具有自主知識產(chǎn)權(quán)的基因資源,課題研究具有較為重要的理論和實踐意義。主要實驗結(jié)果如下： 1、以接種甘蔗黑穗病菌48h和接種無菌水48h時間點的蔗芽為材料,利用抑制消減雜交技術(shù),構(gòu)建黑穗病菌脅迫下的甘蔗正、反向SSH文庫。隨后從文庫中隨機挑取24個克隆進(jìn)行PCR鑒定,結(jié)果顯示插入片段長度主要分布于150～750bp之間,說明本研究構(gòu)建的SSH文庫質(zhì)量良好。 2、從SSH文庫中,篩選768個陽性克隆,進(jìn)行反向Northern雜交,經(jīng)過分析雜交信號強度,篩選獲得190個差異表達(dá)基因。 3、對經(jīng)反向Northern雜交驗證到的190個陽性克隆進(jìn)行測序及生物信息分析,去除重復(fù)、污染及低質(zhì)量(100bp)序列后,獲得了174個差異表達(dá)基因,這些基因主要存在于高分子配體(macromolecular complex)或細(xì)胞器(organelle)中,具有催化活性(catalytic activity)和結(jié)構(gòu)分子活性(structural molecule activity),參與糖酵解(Glycolysis/Gluconeogenesis)、光合作物固碳(Carbon fixation in photosynthetic organisms)、半胱氨酸和蛋氨酸代謝(Cysteine and methionine metabolism)、二羧酸代謝(Glyoxylate and dicarboxylate metabolism)、氨基糖和核苷酸糖代謝(Amino sugar and nucleotide sugar metabolism)、氧化磷酸化(Oxidative phosphorylation)等代謝途徑。 4、在差異表達(dá)基因測序及生物信息學(xué)分析的基礎(chǔ)上,開展如下工作： 首先,從SSH文庫中挑選6個差異表達(dá)EST序列,經(jīng)電子克隆和RT-PCR擴增驗證獲得6個差異表達(dá)基因的全長cDNA序列,分別命名為細(xì)胞色素b5還原酶基因(Scb5R,GenBank登錄號：KJ577591)、14-3-3蛋白基因(Sc14-3-3, GenBank登錄號：KJ577592)、乙醇脫氫酶基因(ScADH, GenBank登錄號：KJ577593)、泛素結(jié)合酶基因(ScUBc E2, GenBank登錄號：KJ577594)、真核翻譯起始因子5A基因(SceIF5A, GenBank登錄號：KJ577595)、S-腺苷甲硫氨酸合成酶基因(ScSAM, GenBank登錄號：KJ577596)； 其次,對這6個基因進(jìn)行生物信息學(xué)分析：甘蔗Scb5R基因全長1257bp,ORF長840bp,編碼氨基酸297個,編碼蛋白是一種無信號肽,定位于內(nèi)質(zhì)網(wǎng)的穩(wěn)定、親水、堿性、非分泌蛋白,蛋白質(zhì)二級結(jié)構(gòu)主要為無規(guī)則卷曲,主要功能為轉(zhuǎn)運結(jié)合；甘蔗Sc14-3-3基因全長1048bp, ORF長771bp,編碼氨基酸256個,編碼蛋白是一種無信號肽,定位于細(xì)胞質(zhì)的穩(wěn)定、親水、酸性、非分泌蛋白,蛋白質(zhì)二級結(jié)構(gòu)主要為α-螺旋,主要功能為翻譯和能量代謝；甘蔗ScADH基因全長1644bp, ORF長1140bp,編碼氨基酸379個,編碼蛋白是一種無信號肽,定位于葉綠體基質(zhì)的穩(wěn)定、親水、酸性、非分泌蛋白,蛋白質(zhì)二級結(jié)構(gòu)主要為無規(guī)則卷曲,主要功能為能量代謝和翻譯；甘蔗ScUBc E2基因全長997bp, ORF長447bp,編碼氨基酸148個,編碼一種無信號肽,定位于細(xì)胞質(zhì)的不穩(wěn)定、親水、堿性、非分泌蛋白,蛋白質(zhì)二級結(jié)構(gòu)主要為無規(guī)則卷曲；甘蔗SceIF5A基因全長1174bp, ORF長483bp,編碼氨基酸160個,編碼蛋白是一種無信號肽,定位于細(xì)胞質(zhì)的穩(wěn)定、親水、酸性、非分泌蛋白,蛋白質(zhì)二級結(jié)構(gòu)主要為無規(guī)則卷曲,主要功能為翻譯和能量代謝；甘蔗ScSAM基因全長1788bp, ORF長1196bp,編碼氨基酸396個,是一種無信號肽,定位于細(xì)胞質(zhì)的穩(wěn)定、親水、酸性、非分泌蛋白,蛋白質(zhì)二級結(jié)構(gòu)主要為無規(guī)則卷曲,主要功能為能量代謝和翻譯。 最后,通過實時熒光定量PCR技術(shù),對這6個基因在葉片、側(cè)芽、蔗髓和根等不同組織中的表達(dá)情況及其在黑穗病菌、SA、MeJA和ABA脅迫下的表達(dá)特征進(jìn)行分析,結(jié)果顯示：這6個基因的轉(zhuǎn)錄本,除甘蔗Sc14-3-3基因、ScADH基因外,其余均在側(cè)芽中檢測到最高的轉(zhuǎn)錄水平。在品種YC05-179中,甘蔗Scb5R基因在受黑穗病菌脅迫及SA、MeJA和ABA誘導(dǎo)后表達(dá)均呈“揚-抑-揚”的趨勢；甘蔗Sc14-3-3基因的表達(dá)受到黑穗病菌、SA、MeJA和ABA強烈誘導(dǎo)表達(dá)；甘蔗ScADH基因受ABA誘導(dǎo)表達(dá),同時受黑穗病菌先抑制后誘導(dǎo)表達(dá),SA和MeJA先誘導(dǎo)后抑制表達(dá)；甘蔗ScUBc E2基因受SA和MeJA誘導(dǎo)表達(dá)。甘蔗SceIF5A基因受黑穗病菌、SA、MeJA和ABA誘導(dǎo)表達(dá)；甘蔗ScSAM基因在接種黑穗病菌后呈“揚-抑-揚”表達(dá)趨勢,受SA誘導(dǎo)后,呈“揚-抑”趨勢。
[Abstract]:Sugarcane smut is an airborne fungal disease caused by Sporisorium scitamineum. It has become one of the major diseases in sugarcane planting areas all over the world. Sugarcane smut can reduce the yield and sugar content of sugarcane and cause serious losses to sugarcane industry. It is also the most important way, however, because the resistance genes of sugarcane to Smut mostly come from wild species, and the high-yield and high-sugar genes in wild species are closely linked with some unfavorable genes, so the traditional sugarcane hybrid breeding has introduced some unfavorable non-target genes as well as disease-resistant genes. Based on the analysis of the molecular mechanism of interaction between sugarcane and smut fungus, the key genes for disease resistance were excavated, and then these genes were transfected into the target sugarcane varieties by transgenic means to achieve the goal of directional improvement of sugarcane resistance. In this study, sugarcane variety ROC22 was used as experimental material. First, suppression subtractive hybridization (SSH) was used to construct a cDNA library. Second, reverse Northern hybridization was used to construct the library. Finally, on the basis of bioinformatics analysis of the cloned genes, tissue-specific expression of these genes and SA (salicylic acid) were detected by real-time fluorescence quantitative PCR. MeJA (methyl-jasmonate) and ABA (abscisic acid) stress expression characteristics, in order to reveal the role of these candidate genes in the resistance of sugarcane to Smut mechanism, for the follow-up use of sugarcane genetic engineering means to improve the directional resistance of sugarcane varieties to smut, to provide gene resources with independent intellectual property rights, the research has more. This is an important theoretical and practical significance. The main results are as follows:
1. Sugarcane SSH libraries were constructed by suppression subtractive hybridization (SSH) using sugarcane buds inoculated with smut fungus for 48 h and sterile water for 48 h. 24 clones were randomly selected from the library for PCR identification. The results showed that the inserted fragments were mainly distributed between 150 and 750 bp, indicating that the SSH libraries of Sugarcane under smut stress were constructed. The constructed SSH library is of good quality.
2. 768 positive clones were screened from SSH library, and 190 differentially expressed genes were obtained by reverse Northern hybridization.
3. Sequencing and bioinformatics analysis of 190 positive clones verified by reverse Northern hybridization showed that 174 differentially expressed genes were obtained after removing repetitive, contaminated and low-quality (100 bp) sequences. These genes were mainly found in macromolecular complexes or organelles with catalytic activity. Activity and structural molecule activity are involved in Glycolysis / Gluconeogenesis, Carbon fixation in photosynthetic organisms, Cysteine and methionine metabolism, Glyoxylate and dicarboxylate metabolism, and aminoglycose metabolism. And nucleotide metabolism, oxidative phosphorylation and other metabolic pathways.
4, based on the sequencing of differentially expressed genes and bioinformatics analysis, the following work is carried out:
Firstly, six differentially expressed EST sequences were selected from SSH library, and the full-length cDNA sequences of six differentially expressed genes were obtained by electronic cloning and RT-PCR amplification. They were named as cytochrome b5 reductase gene (Scb5R, GenBank login number: KJ577591), 14-3-3 protein gene (Sc14-3-3, GenBank login number: KJ577592), ethanol dehydrogenase gene (S CADH, GenBank login number: KJ577593, ubiquitin-binding enzyme gene (ScUBc E2, GenBank login number: KJ577594), Eukaryotic translation initiation factor 5A gene (SceIF5A, GenBank login number: KJ577595), S-adenosylmethionine synthase gene (ScSAM, GenBank login number: KJ577596);
Secondly, bioinformatics analysis of the six genes was carried out: the sugarcane Scb5R gene was 1257 BP in length, the ORF was 840 BP in length and encoded 297 amino acids. The encoded protein was a non-signaling peptide, located in the stable, hydrophilic, alkaline and non-secretory endoplasmic reticulum. The secondary structure of the protein was mainly irregular curl, and the main function was transport binding. The total length of the gene is 1048 bp, the ORF is 771 bp, encoding 256 amino acids. The encoded protein is a non-signaling peptide, located in the cytoplasm of stable, hydrophilic, acidic, non-secretory protein. The secondary structure of the protein is mainly alpha-helix, and the main function is translation and energy metabolism. Protein is a non-signaling peptide, located in the stability of chloroplast matrix, hydrophilic, acidic, non-secretory protein, protein secondary structure is mainly irregular curl, the main function is energy metabolism and translation; sugarcane ScUBc E2 gene 997 bp, ORF 447 bp, coding amino acids 148, coding a non-signaling peptide, located in the cytoplasm of the instability SceIF5A gene of sugarcane is 1174 BP long, ORF is 483 BP long, and encodes 160 amino acids. The encoded protein is a non-signaling peptide, located in the cytoplasm of stable, hydrophilic, acidic, non-secreting proteins. The secondary structure of protein is mainly irregular curl, and the main function is as follows: Translations and energy metabolism; sugarcane ScSAM gene is 1788 BP long, ORF 1196 BP long, encoding 396 amino acids, is a signal-free peptide, located in the cytoplasm of stable, hydrophilic, acidic, non-secretory protein, protein secondary structure is mainly irregular curl, the main function of energy metabolism and translation.
Finally, the expression of these six genes in leaf, lateral bud, sugarcane pulp and root tissues and their expression characteristics under SA, MeJA and ABA stress were analyzed by real-time fluorescence quantitative PCR. The results showed that the transcripts of these six genes, except for Sc14-3-3 gene and ScADH gene, were detected in lateral buds of sugarcane. In YC05-179, the expression of Scb5R gene in sugarcane showed a trend of "rising-inhibiting-rising" under the stress of Smut Fungus and SA, MeJA and ABA, the expression of Sc14-3-3 gene in sugarcane was strongly induced by Smut Fungus, SA, MeJA and ABA, and the expression of ScADH gene in sugarcane was induced by ABA and inhibited by Smut Fungus at the same time. Sugarcane ScUBc E2 gene was induced by SA and MeJA, sugarcane SceIF5A gene was induced by Smut Fungus, SA, MeJA and ABA, and sugarcane ScSAM gene was expressed in a trend of "rising-inhibiting-raising" after inoculation with Smut fungus.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：S435.661;Q943.2
本文編號：2221920
[Abstract]:Sugarcane smut is an airborne fungal disease caused by Sporisorium scitamineum. It has become one of the major diseases in sugarcane planting areas all over the world. Sugarcane smut can reduce the yield and sugar content of sugarcane and cause serious losses to sugarcane industry. It is also the most important way, however, because the resistance genes of sugarcane to Smut mostly come from wild species, and the high-yield and high-sugar genes in wild species are closely linked with some unfavorable genes, so the traditional sugarcane hybrid breeding has introduced some unfavorable non-target genes as well as disease-resistant genes. Based on the analysis of the molecular mechanism of interaction between sugarcane and smut fungus, the key genes for disease resistance were excavated, and then these genes were transfected into the target sugarcane varieties by transgenic means to achieve the goal of directional improvement of sugarcane resistance. In this study, sugarcane variety ROC22 was used as experimental material. First, suppression subtractive hybridization (SSH) was used to construct a cDNA library. Second, reverse Northern hybridization was used to construct the library. Finally, on the basis of bioinformatics analysis of the cloned genes, tissue-specific expression of these genes and SA (salicylic acid) were detected by real-time fluorescence quantitative PCR. MeJA (methyl-jasmonate) and ABA (abscisic acid) stress expression characteristics, in order to reveal the role of these candidate genes in the resistance of sugarcane to Smut mechanism, for the follow-up use of sugarcane genetic engineering means to improve the directional resistance of sugarcane varieties to smut, to provide gene resources with independent intellectual property rights, the research has more. This is an important theoretical and practical significance. The main results are as follows:
1. Sugarcane SSH libraries were constructed by suppression subtractive hybridization (SSH) using sugarcane buds inoculated with smut fungus for 48 h and sterile water for 48 h. 24 clones were randomly selected from the library for PCR identification. The results showed that the inserted fragments were mainly distributed between 150 and 750 bp, indicating that the SSH libraries of Sugarcane under smut stress were constructed. The constructed SSH library is of good quality.
2. 768 positive clones were screened from SSH library, and 190 differentially expressed genes were obtained by reverse Northern hybridization.
3. Sequencing and bioinformatics analysis of 190 positive clones verified by reverse Northern hybridization showed that 174 differentially expressed genes were obtained after removing repetitive, contaminated and low-quality (100 bp) sequences. These genes were mainly found in macromolecular complexes or organelles with catalytic activity. Activity and structural molecule activity are involved in Glycolysis / Gluconeogenesis, Carbon fixation in photosynthetic organisms, Cysteine and methionine metabolism, Glyoxylate and dicarboxylate metabolism, and aminoglycose metabolism. And nucleotide metabolism, oxidative phosphorylation and other metabolic pathways.
4, based on the sequencing of differentially expressed genes and bioinformatics analysis, the following work is carried out:
Firstly, six differentially expressed EST sequences were selected from SSH library, and the full-length cDNA sequences of six differentially expressed genes were obtained by electronic cloning and RT-PCR amplification. They were named as cytochrome b5 reductase gene (Scb5R, GenBank login number: KJ577591), 14-3-3 protein gene (Sc14-3-3, GenBank login number: KJ577592), ethanol dehydrogenase gene (S CADH, GenBank login number: KJ577593, ubiquitin-binding enzyme gene (ScUBc E2, GenBank login number: KJ577594), Eukaryotic translation initiation factor 5A gene (SceIF5A, GenBank login number: KJ577595), S-adenosylmethionine synthase gene (ScSAM, GenBank login number: KJ577596);
Secondly, bioinformatics analysis of the six genes was carried out: the sugarcane Scb5R gene was 1257 BP in length, the ORF was 840 BP in length and encoded 297 amino acids. The encoded protein was a non-signaling peptide, located in the stable, hydrophilic, alkaline and non-secretory endoplasmic reticulum. The secondary structure of the protein was mainly irregular curl, and the main function was transport binding. The total length of the gene is 1048 bp, the ORF is 771 bp, encoding 256 amino acids. The encoded protein is a non-signaling peptide, located in the cytoplasm of stable, hydrophilic, acidic, non-secretory protein. The secondary structure of the protein is mainly alpha-helix, and the main function is translation and energy metabolism. Protein is a non-signaling peptide, located in the stability of chloroplast matrix, hydrophilic, acidic, non-secretory protein, protein secondary structure is mainly irregular curl, the main function is energy metabolism and translation; sugarcane ScUBc E2 gene 997 bp, ORF 447 bp, coding amino acids 148, coding a non-signaling peptide, located in the cytoplasm of the instability SceIF5A gene of sugarcane is 1174 BP long, ORF is 483 BP long, and encodes 160 amino acids. The encoded protein is a non-signaling peptide, located in the cytoplasm of stable, hydrophilic, acidic, non-secreting proteins. The secondary structure of protein is mainly irregular curl, and the main function is as follows: Translations and energy metabolism; sugarcane ScSAM gene is 1788 BP long, ORF 1196 BP long, encoding 396 amino acids, is a signal-free peptide, located in the cytoplasm of stable, hydrophilic, acidic, non-secretory protein, protein secondary structure is mainly irregular curl, the main function of energy metabolism and translation.
Finally, the expression of these six genes in leaf, lateral bud, sugarcane pulp and root tissues and their expression characteristics under SA, MeJA and ABA stress were analyzed by real-time fluorescence quantitative PCR. The results showed that the transcripts of these six genes, except for Sc14-3-3 gene and ScADH gene, were detected in lateral buds of sugarcane. In YC05-179, the expression of Scb5R gene in sugarcane showed a trend of "rising-inhibiting-rising" under the stress of Smut Fungus and SA, MeJA and ABA, the expression of Sc14-3-3 gene in sugarcane was strongly induced by Smut Fungus, SA, MeJA and ABA, and the expression of ScADH gene in sugarcane was induced by ABA and inhibited by Smut Fungus at the same time. Sugarcane ScUBc E2 gene was induced by SA and MeJA, sugarcane SceIF5A gene was induced by Smut Fungus, SA, MeJA and ABA, and sugarcane ScSAM gene was expressed in a trend of "rising-inhibiting-raising" after inoculation with Smut fungus.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：S435.661;Q943.2
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