Study On Cloning Of HDS Gene And Resistant Transcription Factor Of Narcissus Tazetta Linn.

Narcissus tazetta Linn.is an important member of the Amaryllidaceae superfamily,belonging to the monocotyledonous bulbous flowers.Narcissus tazett var is one of China’s ten traditional flowers,with a high ornamental value and medicinal value,mainly from Fujian,Shanghai,Zhejiang and Jiangsu coastal area.The Narcissus tazetta Linn.main cultivars is relatively simple,mainly in the Jinzhanyintai and Yulinglong.Narcissus tazetta Linn.’s germplasm resource is very scarce,because Narcissus tazetta Linn.is autotriploid.It is difficult to through traditional breeding to improved varieties,in largely limits the Narcissus tazetta Linn.breeding work smoothly.And because of Narcissus tazetta Linn.’s long-term asexual reproduction and virus accumulation,resulting in its resistance to weaken,the variety of degradation,flower pavilion and flower number reduced,fragrance fades,which seriously restricted the development of China’s Narcissus tazetta Linn.industry.Therefore,the use of molecular biology and genetic engineering methods to improve Narcissus tazetta Linn.varieties has become a new effective breeding way,which has a good prospect for the improvement of floral fragrance and resistance.The main research contents and results are as follows:1,The extraction of Jinzhantintai,Huanghua Ⅱ and Baihua II respectively coated petals and deputy crown bud period,RNA,and reverse transcription into cDNA.According to the previous He yan-sen Narcissus tazetta Linn.subtractive library of HDS gene segments of conservative district,were cloned by RT-PCR and RACE technology HDS gene in the three different varieties of narcissus:NtHDSY,NtHDSJ and NtHDSW and sequence analysis,the result of the real-time fluorescence quantitative analysis shows that in full bloom,NtHDS gene expression quantity is higher than the bud period,shows that coated synthesis of a large number of terpenoid substances are needed to attract and guide the insect pollination,thus ensuring the genetic diversity and stability of the plant groups.2.Preliminary selected from Yunxiang database of the transcriptome NAC gene family of seven,with these gene fragment sequence to design fluorescent quantitative primer,with concentration of 100 umol/L ABA stress with burgeoning bulb,extracting leaf RNA as template,and reverse transcription by fluorescence quantitative PCR results showed that numbers for CL3249.Contig3 and CL4883.Contig3 two NAC genes expression of significant rise in quantity,so you could choose these two genes as objective to further study.3,According to the selected two NAC genes in the transcriptome fragment sequences in the database to design the specific primers,respectively using conventional PCR technology related to resistance gene in the cloned Yunxiang NtNACa and NtNACb.By using the primers of NtNACa gene,the NtNACah gene of Huanghua Ⅱ was cloned and made the homology comparison.Then these three NtNAC genes were cloned and did bioinformatics analysis.Homology of Yunxiang NAC gene family in two different NAC genes and other plant NAC genes are highly conserved and different types of Narcissus tazetta Linn.varieties Yunxiang and Huanghua Ⅱ in the same NAC genes homology of the difference is very small.Subcellular localization showed that these three NAC genes were located in the nucleus,which consistent with the research results of Dr.Lumin and Dr.Zhu zi-guo.4,The expression of NtNACa and NtNACb genes in different tissues as well as in plant growth regulator ABA,high temperature,PEG400 and different concentrations of NaCl were analyzed by fluorescence quantitative results.Tissue specific expression analysis found that the expression of the two genes were highest in the leaves,the root of the second,and the lowest in the bulb.Different stress treatment results showed that the two genes were more sensitive to ABA stress than to NaCl,PEG400 and high temperature stress.Under 24h short-term stress treatment time,the expression of NAC gene in the leaves was highest at third hour under most treatment conditions.The expression level of the two genes in the root was more significant than that in the leaves under the same treatment.5,The use of cloned NtNACa and NtNACb gene,recombinant plasmid was constructed respectively pBI301-NtNACa-GUS and pBI1301-NtNACb-GUS,lay good foundation for the later gm research.