Cloning And Differential Expression Analysis Of Genes Related To Vernalization And Photoperiod Regulation In Tall Fescue

Tall fescue is the major cool-season perennial turfgrass with excellent properties, such as drought-resistance, barren-resistance, strong disease-resistance, abroad adaptability, and become one of the major turfgrass. So, it widely used in construction of urban open space and sports fields. However, the stems that start to emerge during the growth season suppress the formation of new shoots and affect the quality. Therefore, the inhibition of floral transition and the expansion of vegetative phase have become new breeding ways of genetic improvemen by studying molecular characteristics of genes related to vernalization and photoperiod regulation in tall fescue.The study of cloning and molecular characteristics of gene in tall fescue are very little. In this paper, we reported molecular characteristics, differential expression of vernalizational gene FaVRN1, photoperiodic regulation gene FaCONSTANS, SVP-like gene FaVRT-2, S-adenosylme-thionine decarboxylase FaSANDC by using homologous amplification, rapid amplification of cDNA ends, real-time fluorescence quantitative PCR and subcellular localization of green fluorescent protein techniques. RNA interference expression vectors were constructed by using designed siRNA from FaVRN1 and FaCONSTANS gene sequence.Molecular characteristics of genes related to vernalization and photoperiod regulationThe cDNA of FaVRN1 gene is 1222 bp in length and contains a single open reading frame (ORF,152～889 bp), encodes a protein of 245 amino acids. It has two conserved domains of MADS-box, K-box and many phosphorylation sites. It shows extensive similarities to the known VRN1 of gramineous plants, amino acids sequence of VRN1 was more than 90%.The cDNA of FaCONSTANS gene is 1557 bp in length and contains a single open reading frame (ORF,166～1296 bp), encodes a protein of 376 amino acids. It have zinc finger B-boxes near the amino terminus, and a CCT (CO, CO-like, TOC1) domain near the carboxy terminus. B-box1 was well conserved, B-box2 was not well conserved and lacked three highly conserved C residues,The cDNA of FaVRT-2 gene is 1111 bp in length and contains a single open reading frame (ORF,114-794 bp), encodes a putative protein of 226 amino acids with a conserved MADS (M) domain, a Keratin-like (K) domain and a C-terminal (C) domain. The phylogenetic relationship shows that FaVRT-2, the closest relationship with VRT-2 protein from ryegrass, barley, and wheat, belongs to StMADS11 subfamily of MADS-box transcription factors.The cDNA of FaSAMDC is 1964 bp in length and contains three open reading frames, tiny upstream ORF (226bp-234 bp), small upstream ORF (234～380 bp), main ORF (555-1742bp) respectively. Main ORF encodes a protein of 395 amino acids. Domain analysis reveals that proteins encoded by FaSAMDC genes contain two conserved domains: proenzyme splice site domains and involving SAMDC protein rapid degradation PEST domain. FaSAMDC showed a close genetic relationship with the known SAMDC of monocotyledonous plantsDifferential expression of genes related to vernalization and photoperiod regulationExpression level of FaVRNl gene under vernalization condition increased with prolonged processing time, and reduced under non-vernalization condition, FaVRN1 gene expression level under vernalization conditions was much higher than that of non-vernalization conditions. This indicates that FaVRN1 gene is positively regulated by vernalization.A expression peak of FaVRT-2 appeared in both vernalization and nonvernalizaiton conditions which were from low to high and a followed fall. However, FaVRT-2 expression peak under vernalization condition is earlier 14 days than that of nonvernalization condition. This indicates that FaVRT-2 is responsive to vernalizaiton.A expression peak was detected in both long days and short days condition. Expression peak of FaCONSTANS gene occurs in light under long-days condition,express-ion peak of FaCONSTANS gene occurs in the dark under short-day condition.However, the level of FaCONSTANS transcription peak was significantly higher (2.3 fold) under short days than nuder long days, the peak of FaCONSTANS transcription level under short days condition is earlier 2 hours than that of long days condition. This indicates that FaCONSTANS gene expressed with a circadian rhythm and is regulated by photoperiod.Three expression peaks was detected in both long days and short days condition, The level of FaSAMDC expression peak under long days was significantly higher than that under short days. However, the peak of FaSAMDC transcription level under short days condition is earlier several hours than that of long days condition. This indicates that FaSAMDC gene transcripted with a circadian rhythm and is regulated by photoperiod.Subcellular localizationThree plant expression vectors p-FaCONSTANS-hGFP, p-FaCONSTANS-hGFP and p-FaVRT-2-hGFP with a green fluorescent protein gene has been constructed and was transformed into onion skin cells by gene gun method. The result of transient expression showed that FaVRNl, FaCONSTANS and FaVRT-2 protein was located in the nucleus which is in line with the characteristics of transcription factors.Construction of RNA interference vectorA 351 bp cDNA sequence fragment of FaVRNl gene and FaCONSTANS gene was chosen to construct RNAi expression cassette.Two pairs of special primers with restriction sites was designed according to conservative region of FaVRN1 and FaCONSTANS gene. The conserved fragment of FaVRN1 and FaCONSTANS was amplified by RT-PCR. The amplified fragment was inserted into the sides of intron of middle expression vector pBI121-M-INT in positive and negative orientation after appropriate restriction enzyme digestion. So, the RNAi expression vector with hairpin RNA structures had been constructed.These results suggest that vernalization gene FaVRN1, photoperiod regulation gene FaCONSTANS, SVP-like gene FaVRT-2 and S-adenosylmethionine decarboxylase gene FaSANDC were related to vernalization and photoperiod. We expected to regulated these genes expression level and explore new molecular breeding ways of inhibition for floral transition in tall fescue, So, further study will be required to reveals their molecular regulation mechanism in floral transition,...