Cloning Of DWARF14 Gene From Daylily(Hemerocallis Spp.) And Exploration Of Its Genetic Transformation Conditions

Daylily（Hemerocallis spp.）is a perennial flower of daylily in the family of Aphoraceae,which has a long history of cultivation in China.The branching of scape of daylily affects the yield and ornamental characters of daylily.The development of plant scape/inflorescence is a complex biological phenomenon,which is not only regulated by endogenous genes,but also affected by many factors such as external environment,among which flowering genes and plant hormones play a key role.Strigolactones（SLs）is a new plant hormone,which was first isolated from the secretion of cotton roots.SL can stimulate seed germination and effectively control plant branching and tillering.The research shows that the growth of tiller buds can be regulated by the Max/RMS/D pathway of diopside in rice,which may be conservative in higher plants.DWARF14（D14）gene is one of the genes related to the signal transduction pathway of Unipodophyllide,which can encodeα/βHydrolase protein can not only act as a receptor protein,but also respond to unipodophyllolactone and form bioactive products.Therefore,it is of great significance to study the regulation of SLs in plant scape/inflorescence branching.Based on the transcriptome data of’Autumn Red’,this study designed5 pairs of primers,including Hemerocallis spp.varieties,’Black Eyed Stella’,’Stella de Oro’,’Orange Fairy’and’Thumb Fairy’;Hemerocallis citrina varieties,’Dingzhuang Dacai’,’Jingzhou Flower’,’Datong Flower’7 varieties as the research object.The D14 gene sequence of different varieties of daylily was analyzed and correlated with the character of scape branching of daylily.The study provided a new idea for the regulation of scape branching and plant type of daylily.The main research contents are as follows:（1）During the flowering period of daylily,the investigation was conducted at the National Germplasm Resource Bank of Daylily of Shanghai Institute of Technology on the branching of scape of eight varieties,including’Black Eyed Stella’,’Stella de Oro’,’Orange Fairy’,’Autumn Red’,’Thumb Fairy’,’Dingzhuang Dacai’,’Jingzhou Flower’,and’Datong Flower’.The results showed that the scape branches of eight varieties of daylily belonged to three types:upper inflorescence,8-branch inflorescence and 3-branch inflorescence.There was a signifi cant difference in the number of flower buds on the branches between Hemerocallis spp.and Hemerocallis citrina.The number of flower buds on the branches of Hemerocallis citrina was significantly higher than that of Hemerocallis spp.,but the number of branches and the total number of flower buds showed a dominant positive correlation.（2）In this study,five D14 gene specific primers were designed to amplify the D14 gene of seven varieties including’Black Eyed Stella’,’Stella de Oro’,’Orange Fairy’,’Autumn Red’,’Thumb Fairy’,’Dingzhuang Dacai’,’Jingzhou Flower’,and’Datong Flower’.The relevant bioinformatics analysis was carried out with the’Autumn Red’D14 gene as the control.The results showed that the total length of the amplified gene CDS was about 831 bp.According to the prediction,D14 protein is different in hydrophobicity,but it belongs to hydrophobic protein.The prediction of physical and chemical properties shows that there are differences in their isoelectric point and secondary p rotein structure prediction,which may lead to differences in protein structure.The prediction of tertiary protein structure further confirmed this point.（3）In this experiment,the fresh seeds of daylily’Black Eyed Stella’were used for callus induction and a large number of calli were obtained.Agrobacterium-mediated transformation of daylily callus and plant was carried out.The results show that the optimum medium for callus growth was MS+2 mg/L BA+1 mg/L NAA+0.3 mg/L 2,4-D.The suitable conditions for the genetic transformation of daylily callus were as follows:the agrobacterium OD ₆₀₀ value was 0.8,the infection time should be controlled at 10 min,the concentration of 0.2 mmol/L AS was used to co-culture for 3 days,and 300 mg/L Termetine was used to inhibit bacteria for3-5 min;The suitable condition for genetic transformation of y oung leaves of daylily is the agrobacterium OD₆₀₀ value of 0.8,and the co-culture time should be controlled within 3 days.