基因枪介导的高羊茅基因转化体系的建立

用高羊茅 (FestucaarundinaceaSchreb .)种胚离体诱导的胚性愈伤组织作受体进行了基因枪的轰击试验 ,确定了以轰击 2枪 ,子弹距可裂圆片 6cm ,子弹距轰击受体 6cm为最佳轰击参数 ,建立了基因枪优化转化方案 ,获得了遗传稳定的抗除草剂PPT的转化植株。随机抽取的转化植株的Bar基因PCR检测、总DNA点杂交 ,皆成阳性 ,除草剂PPT抗性试验表明 ,转化植株比对照有了更强的抗性。

高羊茅光周期调控基因CONSTANS的克隆与分析

本研究以坪用型"黔草1号"高羊茅为实验材料,基于多年生黑麦草CONSTANS(CO)基因序列设计引物,利用3'RACE和5'RACE方法扩增CO基因。结果表明,从高羊茅中分离出CO基因,命名为FaCON-STANS。经序列分析,cDNA全长1557bp,编码376个氨基酸,具有完整的开放阅读框(ORF,166～1296bp),氨基端具有B-box型锌指结构域,碳端具有CCT(CO,CO-like,TOC1)结构域。进一步的同源性分析表明,Fa-CONSTANS蛋白与禾本科植物的CO蛋白亲缘关系较近,与其它植物的亲缘关系较远。

Cloning and Expression Analysis of FaSRP Gene in Festuca arundinacea under Abiotic Stresses

[Objective] This study aimed to study the structure and functions of SRP gene and variation in its expression under abiotic stresses. [Method] Using the SRP sequence obtained from transcriptome sequencing as the template, the full-length cDNA sequence of SRP gene in Festuca arundinacea was amplified using the 3＇RACE and 5＇RACE methods. [Result] The cDNA sequence of SRP gene has a full length of 1 165 bp, and it contains an open reading frame in full length of 855 bp. The encoded protein by SRP gene is composed of 284 amino acids, and contains a REF domain. The bioinformatic researches on structures and functions of SRPs show that the SRP gene in Festuca arundinacea （FaSRP） has relatively high ho- mologies with SRPs in monocots. Under low nitrogen, drought, high temperature and high salt stresses, the variations in expression of FaSRP gene were studied using fluorescence quantitative PCR. The results showed that FaSRP gene makes responses to low nitrogen, drought and high temperature stresses, but the relevant response mechanisms are not the same, indicating different pathways regulating re- sistance of plants. The expression of FaSRP gene is insensitive to high salt stress. [Conclusion] This study will provide certain candidate gene and technical reserve for breeding of drought- and high temperature-tolerant, nutritious and highly efficient Festuca arundinacea cultivars.

Establishment of genetic transformation system via Agrobacterium in tall fescue cultivar

Tall fescue （Festuca arundinacea Schreb.） is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable, and repeatable approach in transforming the grass using Agrobacterium （EHA105）, where β-glucuronidase gene （uidA） was used as a reporter and hygromycin phosphotransferase gene （hyg） as a selectable marker. An effective expression of transgene was observed in transforming 2-month-old calli derived from mature seeds （cv. Bingo） cultured on MS medium supplemented with 9 mg·L^-1 2, 4-D. A two-step solid medium selection with increasing hygromycin concentration （from 30 to 50 mg· L^-1） was used to obtain resistant calli. Transgenic plants have been produced from many independent transformed calli. The presence of functional β-glucuronidase gene （uidA） was detected in hygromycin-resistant calli. Transgenic plants were regenerated and PCR and Southern blot confirmed transgene integration in the tall fescue genome.

Construction of RNAi Expression Vector Targeting FaVRN1 Gene in Tall Fescue

[Objective] This study was to construct RNAi expression vector targeting FaVRN1 gene. [Method] A 145 bp Arabidopsis actin intron was inserted into the expression vector to generate an intermediate vector pBI121-M-INT. And then two pairs of specific primers with enzyme restriction sites spanning a 351 bp cDNA conserved sequence fragment of FaVRN1 gene were designed for RT-PCR to construct RNAi expression cassette. The amplified fragment was inserted forwardly and reversely at two sides of the intron to construct an RNAi expression vector with hairpin structure. [Result] Double enzyme digestion（HindIII＋BamHI） showed the intron had been successfully into the vector pBI121. PCR amplification and double enzyme digestion indicated the success of forward and reverse ligation of target FaVRN1 fragment into the intermediate vector. [Conclusion] This study laid foundation for breeding novel flowering-inhibited tall fescue varieties.

Cloning and Differential Expression Analysis of FaGF14-B and FaGF14-C Genes in Tall Fescue

[Objective] The differential expression analysis was performed for FaGF14- B and FaGF14-C genes in tall fescue so as to provide certain basis for follow-up functional analysis of genes. [Method] The sequence fragments of FaGF14-B and FaGF14-C obtained from reverse transcription were used as templates, and the full- length cDNA sequences of FaGF14-B and FaGF14-C were amplified using the 5＇ RACE use 3＇RACE techniques. They were named as FaGF14-B and FaGF14-C, and used for nucleic acid sequence analysis, encoded protein analysis, protein con- served domain analysis, phylogenetic analysis and differential expression analysis. [Result] The FaGF14-B gene has a full length of 1 548 bp. It has a complete open reading frame （ORF, 449-1 228 bp）, and encodes a protein composed of 261 amino acids. The FaGF14-C gene has a full length of 1 250 bp. It also has a complete open reading frame （ORF, 66-848 bp）, and encodes a protein composed of 261 amino acids. The GF14-B and GF14-C proteins all have a typical domain 14-3-3, and their secondary structures all contain 9 conserved co-helical structures and non-conserved N- and C- terminals. The phylogenetic analysis showed that the FaGF14-B and FaGF14-C from tall rescue have high similarities with GF14 protein from gramineous plants, and they are divided into the same clade with closer ge- netic relationship. The real-time fluorescence quantitative PCR analysis showed that the expression of FaGF14-B and FaGF14-C genes is all sensitive to nitrogen stress. [Conclusion] This study will lay a theoretical basis for further screening of low nitrogen-tolerant genes and breeding of low nitrogen-tolerant grass germplasms.

Expression Analysis of 14-3-3 Gene in Tall Fescue under Several Abiotic Stresses

To study the functions of 14-3-3 gene family in tall fescue, the potential functions of 13 14-3-3 proteins in Arabidopsis were investigated by bioinformatic analysis. Based on the sequences of 14-3-3 genes in tall fescue by transcriptome and proteomic sequencing, the full-length cDNA sequences of 4 14-3-3 genes in tall fescue were obtained. Their sequences were aligned by Clustal W2. The results showed that the genetic relationships between 14-3-3A and 14-3-3D, 14-3-3B and 14-3-3C are closer, and their main structures are very conservative. The changes in expression levels of 14-3-3 genes under low nitrogen, drought, high temperature and high salt stresses were investigated by fluorescence quantitative PCR. The expres- sion level of 14-3-3A makes responses to low nitrogen, drought, high temperature and high salt stresses; the expression levels of other genes also make responses to abiotic stresses in varying degrees, but the relevant response mechanisms are not exactly the same. Therefore, it is speculated that the 14-3-3 gene family regu- lates stress resistance of plants through different pathways, and functional differenti- ation occurs during its evolution.

Cloning and Characterization of Phytochrome A Gene Fa PHYA from Tall Fescue

The sequence fragment of PHYA, obtained from transcriptome sequencing,was used as the template, and the full-length c DNA sequence of PHYA gene in tall fescue was amplified using 3＇RACE and 5＇RACE techniques. The c DNA sequence of PHYA gene has a complete open reading frame（ORF, 293-6 682 bp）, and it encodes a protein composed of 1 129 amino acids. The N-terminal of Fa PHYA is composed of GAF and Phytochrome domains, and its C-terminal contains two repeated PAS domains, one histidine kinase A domain and one histidine kinase-like ATPase domain. The homology analysis showed that the amino acid sequences of Fa PHYA of tall fescue and PHYAs of gramineous plants have higher homologies（85%）, indicating close genetic relationships. However, the homologies between FaPHYA of tall fescue and PHYAs of monocotyledons are lower, indicating far genetic relationships.