Cloning And Functional Analysis Of GA 20-oxidase And GA 2-oxidase Genes In Tomato

Gibberellin (GA), an important plant phytohormone, plays a criticlal role in wholeplant development such as stem and root growth, leaf expansion, flower development,fruit set, and seed germination etco Plants can respond to the outer environmental signalthrough the regulation of GA levels. So GA biosynthesis plays a key role in plant growthand in plant response to the enviroment. In horticulture, people often use chemicalreagents such as CCC or PP333 to control the plant heights. Since the GA biosyntheticpathway has been explored in which most of the enzymes involved has been isolated, itenables us to regulate the expression of target gene in the specific tissues bybiotechnology, thus realize the control of plant growth and development.To date, genes encoding enzymes involved in GA biosynthetic pathway has beenisolated which provide us usful information of the GA function in plant growth anddevelopment. GA 20-oxidase and GA 2-oxidase, belonging to two multi-gene familys, aretwo key enzymes involved in GA biosynthetic pathway. GA 20-oxidase is involved in theformation of bioactive GA₁ and/or GA₄ while GA 2-oxidase, which in turn catalyzes thedeactivation of GA₁ and GA₄. So the production of bioactive GA will be influenced by theexpression of these two enzymes. GA 2-oxidase gene has not been cloned from tomato.And research about the specific function of each GA 2-oxidase in GA 20-oxidase genefamily has not been made. The exploration of the different roles of three GA 20-oxidasegenes and GA 2-oxidase gene played in tomato plant will help us to know how GA affectthe plant growth and development and provide us useful knowledge on the agriculturepractice by reasonably using GA.This study aims to clone and regulate the expression of genes involved in tomato GAbiosynthesis and metabolism. We cloned three tomato GA 20-oxidase genes and GA2-oxidase2 gene, gernerated transgenic plants by RNA interference. The gene function inplant growth and development were exploided by the molecular and morphology analysis.The main results were as follows:1. The full-length cDNA of GA 2-oxidase2 (GA2ox2) was cloned and the sequence datahas been submitted to the GenBank databases under accession number EF017805.The full-length of GA2ox2 cDNA is 1203 bp and contains a complete open reading frame (ORF) of 969 bp, which encoding 322 amino acids.2. Semi-quantitative RT-PCR analysis showed that GA 20-oxdiase and GA 2-oxidase2were constitutively expressed in roots, stems, leaves, flowers and fruits of tomato,whereas the expression levels were different.3. Four plant expression vectors were constructed, which produced the double strand ofRNAs of GA20ox1, GA20ox2, GA20ox3 and GA2ox2 cDNA respectively. A total of85 putative transgenic tomato plants were obtained using the Agrobacterium-mediatedtransformation. The PCR and Southern blot analysis of the putative transgenic plantsshowed that the T-DNA was integrated into the tomato genome, with 1 to 6 copies.4. Semi-quantitative RT-PCR analysis from the transgenic plants showed that the aimedgene expression was decreased in RNAi transgenic plants. When one GA 20ox genewas suppressed, the expression of other two GA20ox genes was not affected, whichindicated that RNAi was specific and efficient.5. The ELISA was used to measure the GA contents of transgenic plants. The resultsindicated that the suppression of GA20ox1 and GA20ox2 were significantly reducedGA content while the suppression of GA20ox3 did not change GA levels dramatically.And the decreased expression of GA2ox2 did not affect the GA biosynthesis in plant.6. The phenotypic characteristics and physiological characteristics of transgenic plantswere measured. The results showed that suppression of GA20ox1 or GA20ox2 resultedin shorter stems, decreased length of internodes, and small dark green leaves whileplants with decreased expression of GA20ox3 had no visible changes on stems andleaves except that it has less and longer lateral roots. Three GA 20-oxidase transgenicplants can all flower and fruit normally. The results indicated that GA 20-oxidasemake function in plant vegetative organs such as roots, stems and leaves and showedlittle influence in plant productive growth.7. Severely GA-deficient mutants of some species have impaired seeds or delayed seedgermination. The seed germinations of three GA 20-oxidase transgenic plants weremeasured. The results indicated that the suppression of GA20ox resulted in an overalltrend of delayed germination and lower seed germination ratio.8. Histological analysis of the stem and leaf was conducted. In GA20ox1 and GA20ox2RNAi transgenic plants leaf anatomical characteristics showed that mesophyll cells inthe leaves are much bigger than those from the wild type plants and the palisade tissue cells were much more densely arranged than that in wild type plants. The thickness ofxylem in stems from GA20ox1 and GA20ox2 RNAi transgenci plants was decreased.The leaf and stem anatomical characteristics of GA20ox3 RNAi transgenic plantswere similar with the control. The results suggested that the suppression of GA20ox1and GA20ox2 regulated cell devision and cell formation in stems and leaves.9. The dwarf plant with thickened leaves usually have higher resistance to abiotic stress.The resistances to drought stress of GA20ox transgenic plants were measured. Andthe results indicated that the GA20ox1 and GA20ox2 RNAi transgenic plantsexhibited increased tolerance and resistance to drought stress.