Expression Analysis And Function Investigation During Pollen Germination Of NF-YB8 And NF-YB13 In Picea Wilsonii

NF-Y transcription factors are one of the important transcription factors which universally exist in plants,animals,and microorganisms.It is a trimer protein which is made of three subunits NF-YA,NF-YB,NF-YC.In recent years,NF-Y family members have been cloned and identified in a variety of plants.What is confirmed is that those NF-Y genes have been involved in plant growth and abiotic stress response process.Based on the early studies on Picea wilsonii,our experiment focuses on the transcriptional activity,expression pattern to study whether NF-YB8 and NF-YB13 participate in the process of pollen germination.The main results were as followed:1.Conservative domain structure and development of tree analysis have been used in this research.The results showed that PwNF-YB8 and PwNF-YB13 contains CBFD domain structure which indicated these proteins belong to the family of NF-Y.In evolutionary PwNF-YB8 and PwNF-YB 13 are close to AtNF-YB8 and AtNF-YB 13 in genetic relationship respectively.2.Tissue specific experiment results show that compared with root,stem and acerola,PwNF YB8 and PwNF-YB13 mainly expressed in pollen.Addtionally,PwNF-YB8 and PwNF-YB13 both response to a variety of abiotic stress and hormone treatment although responsing in different patterns.Under the treatment of ABA,the expression of PwNF-YB8 decreased after rising,while PwNF-YB13 expressing on the contrary trend and the overall expression changed small in quantity.Under salt stress,PwNF-YB8 responsed obviously and rose to eight times after 6 h-treatment.In contrast to PwNF-YB8,PwNF-YB13 responsed relatively weak,the highest expression was no more than 2 times.3.The subcellular localization experiments showed PwNF-YB8 and PwNF-YB13 both expressed in the cell nucleus and cytoplasm.Transcription activity analysis showed PwNF-YB13 has transcriptional activation while PwNF-YB8 not.The transcriptional activation domain of PwNF-YB13 is in C terminal.4.As tissue specificity results showed that compared to the needle stem and root,PwNF-YB8 and PwNF-YB13 expressed mainly in pollen,Real-time quantitative PCR have been used.The results showed that PwNF-YB8,PwNF-YB13 were involved in the normal germination of pollen and can be induced by boron acid and calcium ion.5.By Yeast two hybrid method,we found that PwNF-YB8 can interact with another subunit of NF-Y protein.6.PwNF-YB8 and PwNF-YB13 over-expression vector have been transformed to wild-type Arabidopsis successfully.And the positive lines have been selected and obtained by hygromycin and carboxybenzyl selecting plate.All of the candidate lines have been checked on the level of DNA by PCR technique.7.Expriment of transit expression in pollen has been performed,the results showed that empty carrier transformation has no obvious influence on normal pollen germination and pollen tube elongation process.