Identification and functional characterization of potential thermo-tolerance genes in Solanum tuberosum : 유전자 기능성 스크리닝, 발현억제 및 과발현 방법을 이용한 감자의 (Solanum tuberosum) 고온 스트레스 적응 유전자 기능연구

다국어 초록 (Multilingual Abstract)

Potato (S. tuberosum) is a highly heat sensitive crop; a slight rise from optimal temperature can lead to drastic decline in tuber yield. Acclimatization to elevated temperature often demands transcriptional reprogramming of an array of genes to evoke protection from heat stress. First step towards understanding the thermo-tolerance mechanism is to identify the key genes involved in it. Here yeast functional screen method was used to identify potential thermo-tolerance genes from potato. Two cDNA libraries were constructed from heat stressed potato plants (35°C) after 2 and 48 h treatment to identify genes involved in early and late heat stress responsive genes. Ninety-five potential thermo-tolerance candidate genes were identified from yeast cells expressing different cDNAs based on their ability to survive lethal heat stress condition. Moreover, cross- resistance analysis of these clones indicated 20 genes were responsive to drought, 14 to salt and 11 to both stress; suggesting the functional relevance of these genes in abiotic stress tolerance. To obtain molecular evidence on which genes are functionally important for innate and/or acquired thermo-tolerance in higher plants, knockdown (KD) lines for five potato homologs, including GLP1 (Germin-like protein 1), nsLTP (Non-specific lipid transfer protein), PI-PLC (phosphoinositide-specific phospholipase-c), CHP (Conserved hypothetical protein) and RPL4 (60 S Ribosomal L4/L1 protein) were generated using virus induced gene silencing (VIGS) in Nicotiana benthamiana. Silencing of these genes was proved to be effective; an observation was supported by significant reduction in quantitative abundance of the target gene transcripts in respective lines. Among the genes tested, GLP1-KD lines exhibited a strong hypersensitive phenotype under heat stress. To substantiate the inference from the VIGS study, potato transgenic plants constitutively over-expressing StGLP gene were generated and its performance under heat stress was studied. Three (G9, G12 and G15) StGLP transgenic lines exhibited enhanced thermo-tolerance through H2O2 mediated up-regulation of StCAT, StAPX , StGR and other heat stress responsive genes, StHSP70, StHSP20 and StHSP90. These results confirmed further confirmed inference obtaibed from VIGS study, that H2O2 produced by over-expression of StGLP in the transgenic potato plants triggered the ROS scavenging signaling pathways controlling antioxidant and heat stress responsive genes imparting tolerance to heat stress in transgenic plants. To the best of knowledge, this is the first study to describing the role of GLP1 in association with improved thermo-tolerance any economically important crop using transgenic techniques.

국문 초록 (Abstract)

감자는 고온 스트레스에 민감한 작물이다; 생육적온에서 약간의 온도상승만으로도 감자 수확량에 큰 감소를 야기시킬 수 있다. 고온 스트레스에 대한 적응과정은 많은 수의 유전자 발현의 재조정이 필요한 복잡한 과정이다. 이런 고온 적응 기작에 대한 이해는 핵심유전자의 발굴로부터 시작된다. 감자의 고온 적응성 유전자의 분리를 위하여 효모를 이용한 기능성 탐색기법을 이용하였다. 이를 위해 고온 (35°C) 에 2시간과 48시간 노출된 감자로부터 효모 발현 cDNA 유전자 은행 각각 만들고, 고온 조건에서 기능성 탐색을 거쳐 95개의 후보 유전자를 선발하였다. 이들에 대한 다중 스트레스 저항성 시험에서 건조에 대해서는 20개 유전자, 염 스트레스에는 14개 유전자 그리고 11개 유전자는 염과 건조 및 고온에 대하여 저항성을 보였다. 이는 이들 유전자의 비생물적 스트레스 저항성 관련 가능성을 시사한다. 이들 유전자의 식물에서의 고온 저항성 여부를 알아보기 위하여 GLP1 (Germin-like protein 1), nsLTP (Non-specific lipid transfer protein), PI-PLC (phosphoinositide-specific phospholipase-c), CHP (Conserved hypothetical protein) 그리고 RPL4 (60 S Ribosomal L4/L1 protein) 유전자들에 대해 Nicotiana benthamiana를 이용하여 virus induced gene silencing (VIGS)을 적용하였다. VIGS를 통한 대상 유전자들의 발현억제가 모두 확인 되었으며, GLP1-KD 억제 담배의 경우 심한 고온스트레스 민감성을 나타내었다. 이 결과는 GLP1유전자가 정상 발현상태에서 고온 적응성에 주요한 역할을 수행 할 수 있음을 의미한다. VIGS 결과의 검증을 위하여 StGLP1 유전자의 과발현 감자 형질전환체 3개체 (G9, G12 그리고 G15)를 만들고 이들에 대한 고온 저항성을 조사하였다. StGLP1 형질전환 감자는 고온스트레스에 대한 저항성을 보였으며 이는 H2O2 를 통한 세포의 활성산소 제거 유전자인 StCAT, StAPX, StGR 및 단백질 폴딩 조절 유전자 StHSP70, StHSP20 그리고 StHSP90등 스트레스 관련 유전자의 발현증가를 통해 이루어졌다. 이는 VIGS 결과를 뒷받침하며 StGLP1 유전자의 과발현이 H2O2의 증가와 이를 통한 ROS 관련 신호전달 체계를 활성화하여 항산화 물질 및 고온 스트레스 관련 유전자의 발현 증가를 이끌고 결과적으로 형질전환체의 고온스트레스 저항성을 유기한다. 이 결과는 고온 스트레스 저항성에 GLP1 유전자가 관여한다는 최초의 결과이며, 형질전환을 통한 주요경제 작물의 고온 적응성 증진에 주요한 정보이다.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Materials and Methods 7
• 2.1. Identification and characterization of thermo-tolerance genes from potato heat stressed cDNA library using yeast functional screening 7
• 2.1.1. Plant material and growth conditions 7
• 2.1.2. Stress treatments and isolation of RNA from potato 7
• 2.1.3. Preparation of cDNA from heat stressed S.tubersoum and its size separation 8
• 2.1.4. Yeast strains and identification of functional screening temperature 12
• 2.1.5. Large scale yeast functional screening 12
• 2.1.6. Cross-resistance assays 15
• 2.1.7. Plasmid rescue and back transformation to E.coli 15
• 2.1.8. DNA sequencing and gene annotation 17
• 2.1.9. Validation of yeast functional screen by expression analysis 18
• 2.2. Virus induced gene silencing of selected genes in N. benthamiana and their functional relevance in thermo tolerance 21
• 2.2.1. Growing tobacco (N. benthamiana) plants 21
• 2.2.2. Construction of VIGS plasmids 21
• 2.2.3. Agro-infiltration of tobacco 25
• 2.2.4. Assessing the degree of silencing by quantitative real time PCR 25
• 2.2.5. Imposition of heat stress treatments in N. benthamiana 26
• 2.2.6. Determination of cell membrane stability, cell viability and total chlorophyll content in N. benthamiana VIGS lines 30
• 2.2.7. Determination of oxlate oxidase (OXO) activity and H2O2 content in N. benthamiana VIGS lines 31
• 2.2.8. Antioxidant assays and expression analysis of antioxidant and stress responsive genes in N. benthamiana VIGS lines 32
• 2.2.9. Measurement of IP3 (inositol-1, 4, 5-trisphosphate) content in N. benthamiana VIGS lines 32
• 2.3. Over-expression of germin-like protein to induce thermo-tolerance inpotato 33
• 2.3.1. Isolation and Gateway cloning of StGLP1 gene 33
• 2.3.2. Generation of StGLP1 transgenic potato 34
• 2.3.3. Molecular analysis of StGLP1 potato transgenic plants 36
• 2.3.4. Induction of high-temperature stress on StGLP1 transgenic potato 37
• 2.3.5. Determination of OXO activity and H2O2 content in transgenic potato plants under the influence of inhibitors 38
• 2.3.6. Expression profiling of antioxidant and defense related genes in StGLP1 transgenic potato 38
• 2.3.7. Multiple sequence alignment of StGLP1 protein 39
• 2.3.8. Statistical analysis 40
• 3. Results 42
• 3.1. Identification of thermo-tolerance genes from potato using yeast functional screening 42
• 3.1.1. Optimum heat stress condition for the selection thermo-tolerant yeast clones expressing potato cDNAs 42
• 3.1.2. Outcome of yeast expression library 44
• 3.1.3. Identification and isolation of potential thermo-tolerance genes from S. tuberosum using yeast functional screening 44
• 3.1.4. Computational analysis and functional classification of potato thermo tolerance genes 45
• 3.1.5. Expression pattern of selected genes in potato under heat stress 57
• 3.1.6. Relative tolerance of yeast clones expressing potato cDNAs to heat, drought and salt stress 59
• 3.1.7. Comparison of yeast functional screening with whole potato transcriptome 61
• 3.1.8. Sequence Accession Numbers 61
• 3.2. Functional characterization of potato heat responsive genes in Nicotiana benthamiana through VIGS approach 67
• 3.2.1. Identification of potato high-temperature stress inducible target gene homologs in N. benthamiana 67
• 3.2.2. Effect of VIGS on plant vigor of silenced plants under heat stress 69
• 3.2.3. Effect of gene knock-down of GLP1, nsLTP, PI-PLC, CHP and RP4 genes on thermo-tolerance 70
• 3.2.4. Cell membrane stability of VIGS lines under heat stress conditions 73
• 3.2.5. Chlorophyll maintenance of VIGS lines under heat stress conditions 73
• 3.2.6. Cell viability of VIGS lines under heat stress conditions 74
• 3.2.7. PI-PLC dependent production of IP3 content of VIGS lines under heat stress conditions 75
• 3.2.8. Expression analysis of the nsLTP, GLP1, PI-PLC, CHP and RPL4 genes in VIGS lines under heat stress 77
• 3.2.9. OXO activity and H202 production in GLP1 and nsLTP silenced plants under heat stress 80
• 3.2.10. Antioxidant activity and expression analysis of antioxidant and stress responsive genes in VIGS lines under heat stress 82
• 3.3. Enhanced tolerance of transgenic potato plants over-expressing StGLP1 gene against high-temperature stress 87
• 3.3.1. Sequence analysis of cDNA encoding StGLP1 87
• 3.3.2. Expression pattern of StGLP1 gene in potato plants under heat stress 92
• 3.3.3. Molecular analysis of transgenic potato plants over-expressing StGLP1 gene 93
• 3.3.4. Enhanced tolerance of StGLP1 transgenic plants to heat stress 95
• 3.3.5. Enhanced StGLP1 depenedent-H2O2 signaling in putative transgenic lines of potato under heat stress 95
• 3.3.6. Enhanced antioxidant enzyme activity in StGLP1 transgenic plants 99
• 3.3.7. Expression analysis of antioxidant and heat responsive genes in StGLP1 transgenic plants under heat stress 101
• 4. Discussion 106
• 4.1. Identification of potential heat stress tolerance genes using yeast functional screening approach 106
• 4.1.1. Stress/defense associated genes induced by heat stress 108
• 4.1.2. Heat shock proteins/molecular chaperons 108
• 4.1.3. Metabolism/signal transduction related genes 109
• 4.1.4. Protein turnover 109
• 4.1.5. Transcription factors 110
• 4.1.6. Carbohydrate metabolism 111
• 4.1.7. Genes with unknown functions 111
• 4.1.8. Most important candidate genes for the improvement of thermo-tolerance and other abiotic stress tolerance of potato 112
• 4.2. Functional relevance of nsLTP, GLP1, PI-PLC, CHP and RPL4 genes in imparting thermo-tolerance using VIGS 113
• 4.2.1. Role of PI-PLC gene in inducing basal thermo-tolerance of tobacco 113
• 4.2.2. Role of nsLTP gene for inducing acquired thermo-tolerance of N. benthamiana 115
• 4.2.3. Role of GLP1 gene for inducing basal and acquired thermo-tolerance of N. benthamiana 116
• 4.2.4. Role of CHP and RPL4 gene in thermo-tolerance of tobacco 118
• 4.2.5. A putative model representing the role of GLP1, n