Study On SCoT Marker And Analysis On Genes Of Stress-related And Important Flowering Time In Mango

Mango(Mangifera indica L) is one of the important tropical fruit, known as the king of fruits.There are a long history of cultivation, rich germplasm resources and many varieties or cultivars about mango in China. Because many mango cultivars were selected from naturally occurring open-pollinated seedlings, their parents were unknown. The genetic relationships among main cultivars, cultivars introduced and some local unique cultivars of mango in our country were unclear, which brought a lot of problems for mango germplasm resources collection, protection and utilization. Abiotic stresses often occur by the global climate change, while mango is sensitive to abiotic stress, therefore the abiotic stresses are major environmental factors that influence the quality and yield of mango seriously. Off-season of mango is the right chose to increase yield and benefit at the same time. Mangifera indica L. cv.’Chok Anand’ can blossom several times without low temperature of the year, which is the most suitable for off-season production, while there have been no reports on the molecular mechanism of flowering about Mangifera indica L. cv.’Chok Anand’. Based on the above reasons, SCoT marker technique was firstly used to study the genetic diversity and relationships among mango accessions, cDNA-SCoT technology improved was first applied to isolate and characterize differential genes expressed under stress treated, and full length cloning, bioinformatics, expression patterns and preliminary functions of important flowering time genes were studied in the present study. The main results were as follows:1. In the present study, SCoT markers were first applied to identify the genetic diversity and relationships among mango cultivars.44new SCoT primers were designed, and the total number of SCoT primers amount to80. The usefulness of SCoT versus ISSR markers for identiying the closest mango cultivars was evaluated, the polymorphic percentages amplified by SCoT markers were higher than that of ISSR markers, and the UPGMA cluster result with SCoT markers was better than that with ISSR analysis in their genetic relationships. and mango varieties of the same or near to origin place.could be clustered to the same group by UPGMA cluster with SCoT markers The results demonstrated that the SCoT marker system is very useful in studying genetic diversity and relationship of mango cultivars.2. The cDNA-SCoT technology was improved and applied in differential gene expression analysis of mango under stresses for the first time. The3’end anchor primer was added to cDNA and combined with SCoT primer for PCR amplification. The cDNA-SCoT technology improved is a simple, low-cost, time saving and efficient method for differential gene expression research, and the fragment amplified by cDNA-SCoT is a function gene.37SCoT primers combined with anchor primer were applied to differential genes expression analysis of mango under stresses in the present study,92fragments were successful obtained and most of these genes were first cloned from mango. The40genes among the92fragments were presumed to be transcription factors and the genes of,stress-related protein, growth development and signal transduction.3. The5’end RACE technology was modified and applied in cloning the full length of stress related genes in mango. The main modifications for normal5’end RACE were as followed:Using new reverse transcriptase primer, increasing the concentration of cDNA and tail primer, extending tailing time, simplifying the process, combined with nested PCR and touchdown PCR. The5’ end RACE technology modified was testified to be a new, simple, rapid, high-efficient and low-cost method. In the present study,40full-length genes related with stress were successful obtained by using the51end RACE technology modified.4. Expression analysis of the important stress-related genes in mango. Expression patterns of six stress-responsive functional genes, six transcription factors and four protective enzyme genes were estimated by real-time quantitative PCR, the results showed that these genes were all regulated by low temperture, NaCl and PEG treatments, but there were different expression patterns under the same stress treatment. The transcription factors among the three kinds of stress-related genes were strong response to the stresses.5. Cloning, expression and function analysis Harpin gene of mango. The full length of mango Harpin gene was obtained by cDNA-SCoT technology and RACE technology. The full length of Harpin cDNA is1019bp and contains a complete open reading frame (ORF) of666bp, which encoding222amino acids. The sequence of mango Harpin is no homology with pathogenic bacteria hrp genes. Harpin gene of mango can express in all tissues, and the expression level rise continuely with the fruit development. The expression analysis revealed mango Harpin gene was regulated by low temperature, NaCl, PEG, heavy metal and different treatments after fruit harvest. Sense Harpin vector of mango was successful transformed into tobacco. Sense transgenic plants leaded to early flowering, increase plant height and cause flower structure mutant. More treatments including NaCl, Pb(NO3)2and ZnSO4all did not differ significantly in To generations between transgenic plants and non-transgenic plants. The function of mango Harpin gene could be identified by further studying the T1and T2transgenic plants.6. The full length of mango flowering time genes LFY, CO, SOC1, FT and TFL1were obtained by homology cloning strategy, genome walking and5’end RACE technology modified, and their bio informatics, expression patterns and functions of these genes have been further studied.Bio informatics analysis:The full length of LFY cDNA is1332bp and contains a complete open reading frame (ORF) of1149bp, encoding383amino acids. The LFY protein contains one conserved FLO_LFY superfamily domains. LFY promoter contains several cis-regulatory elements related to stress-induce and light-response, and cis-acting elements involved in ABA-, GA-, CTK-, IAA-and sugar-responsive were also identified in the promoter. The full length of CO cDNA is1141bp and contains a complete open reading frame (ORF) of966bp, encoding322amino acids. The CO protein contains two B-box domains and one CCT domains, belong to the first group of CO gene, and is close to Arabidopsis AtCOL4gene. The full length of SOC1cDNA is841bp and contains a complete open reading frame (ORF) of669bp, encoding223amino acids. The SOC1protein contains one MADS-box domains and one K-box domains, belong to MADS-box gene family. The full length of FT cDNA is781bp and contains a complete open reading frame (ORF) of588bp, encoding196amino acids. The FT protein contains one PEBP domains, belong to FT/TFL1gene family. The full length of TFL1cDNA is721bp and contains a complete open reading frame (ORF) of516bp, encoding172amino acids. The TFL1protein contains one PEBP domains, belong to FT/TFL1gene family.Expression pattern analysis:Real-time-PCR analysis showed that LFY and CO expressed in all vegetative tissues and reproductive organs, but SOC1gene mainly expressed in vegetative tissues. The expression patterns of LFY, CO, SOC1, FT and TFL1in mature leaves, mature stems and young stems in two cultivars of Mangifera indica L. cv.’Chok Anand’and Mangifera indica L. cv.’ZiHua’were almost similar, but also exist obvious difference. The flowering time genes expressed highly during floral induction from November to December, and another peak appeared from March to June in the following year. Whereas the time of peak appeared and the expression level of these genes were different in different tissues at the same time, in the same tissues at different time and in different mango varieties, further analysis showed that mango flowering time genes also existed regulation network.Function analysis:The sense and antisense vector of the five mango flowering time genes were constructed and successful transferred into tobacco, respectively. The copy numbers were differences between transgenic plants, and these flowering time genes normally expressed in the transgenic plants. The plant heights of the To generations of transgenic plants with LFY+、CO+、FT-and TFL-respectively were lower than that of non transgenic plants, but the plant heights of the To generations of transgenic plants with LFY-、CO-、SOC-、FT+and TFL+respectively showed higher. The SOC+transgenic plants did not differ significantly between transgenic plants and non transgenic plants. In the present study, floral organs of transgenic tobacco To generations with mango flowering time genes had happened mutants, but flowering time have not worked as well as had been hoped,. The functions of these genes could be further studied by the T1and T2transgenic plants.