| Transcription regulation is an important mechanism for the regulation of eukaryotic gene expression.It regulates a variety of biological processes,especially transcription factors,and plays an important role in transcription regulation;b ZIP transcription factor is one of the largest and most differentiated gene families found in plant species.It has a wide range of biological functions,for example,promoting the development of seed,flower and early seedling,and participating in hormone signal transduction,etc.The current research on b ZIP has mainly focused on Arabidopsis,rice and corn,with less reports on other plants.Safflower(Carthamus tinctorius L.)is an annual plant of the family Asteraceae,and in great demand.Nevertheless,it is in short supply in Chinese market,due to the difference of planting area environment.Therefore,it is of great significance to study the cultivation of new high-quality safflower strains by genetic means.In this study,bioinformatics was used to screen and identify b ZIP family members from the safflower gene level database,and to predict and verify the expression patterns of safflower b ZIP family genes in different tissues and organs,Screened out two G subfamily genes affected by light:CtbZIP16 and CtbZIP32.The genes were cloned and super-expression vectors were used to infect Arabidopsis thaliana to obtain transgenic plants.The functions of CtbZIP16 and CtbZIP32 genes were further verified and analyzed.The experimental results obtained are as follows:(1)52 highly reliable b ZIP genes were screened from the safflower genome database and named Ctb ZIP1-52;With reference to the classification of Arabidopsis,it is divided into 12 subfamilies,which are A,B,C,D,E,F,G,H,I,J,S,and X subgroups.The sub-family is named as sub-family X,which contains 4 family members;The maximum Ctb ZIP family gene is 86.19 k Da,the smallest is 11.98 k Da,and the isoelectric point ranges from 4.86 to 9.87;according to the average hydrophilicity and hydrophobicity,Ctb ZIP is a hydrophilic protein;A total of 20 Motif were excavated in the Ctb ZIP family of proteins,of which Motif 1 and Motif 2 were identified as b ZIP conserved structures,and Motif 11 was identified as DOG1 conserved structure;most of the Ctb ZIP genes were enriched into two categories of molecular function and biological process;There is an interaction network within the gene family,and a certain cooperative relationship is still maintained between genes.(2)The results of qRT-PCR showed that the expression pattern of the safflower b ZIP gene was basically consistent with the prediction result of the heat map.Most of the members of subgroup A and H had high expression in leaves,and members of subgroup I were mostly in roots.The S subfamily contains a maximum of 17 members,most of which have higher expression levels in the seeds.Two members of the G subfamily,CtbZIP16 and CtbZIP32,are significantly affected by light,and mainly Expressed in seed,cotyledon and hypocotyl tissues.These results indicate that members of the safflower b ZIP family are widely involved in the growth and development of plant tissues or organs.(3)CtbZIP16 and CtbZIP32 genes were successfully cloned,and a plant overexpression vector was successfully constructed to obtain stable genetically inherited T3 generation transgenic seeds.By comparing transgenic Arabidopsis seeds with wild-type Arabidopsis seeds,we found that the transgenic Arabidopsis seeds are light brown,lighter in color,and have a brighter surface and higher light transmittance;the 1000-grain weight of transgenic Arabidopsis seeds is significantly higher than that of wild-type Arabidopsis and the seeds are fuller.By monitoring the germination rate and germination time,the germination time of transgenic Arabidopsis was ahead of wild-type Arabidopsis by more than 24 hours,and the final germination rates were 97.3% and 97.67%.The hypocotyls and roots of transgenic Arabidopsis early seedlings were longer than those of wild-type Arabidopsis thaliana,suggesting that CtbZIP16 and CtbZIP32 genes can promote hypocotyls and root elongation of early seedlings.(4)The expressions of CtbZIP16 and CtbZIP32 genes are affected by light intensity,and they show an upward regulation with the increase of light intensity.At the same time,as the gene expression is increased,the expression levels of RGL2 and PIL5 are significantly reduced,thereby enhancing the role of the GA pathway.These results indicate that CtbZIP16 and CtbZIP32 genes can directly or indirectly regulate the expression of RGL2 and PIL5 genes,downregulate ABA levels and enhance GA levels at the same time,thereby promoting the early development of Arabidopsis. |
