Study On Interacting Protein Of DGP1,A Regulatror For Dark Green Panicle In Rice

In nature,photosynthesis is the source of energy for the growth of all creatures.It can use solar energy to convert inorganic matter into organic matter,which is used by the metabolism of creatures.The production of various food crops is also inseparable from photosynthesis.Only by fully enhancing the photosynthetic capacity and enhancing the photosynthetic efficiency can we effectively increase the output of grain and meet the growing needs of mankind.However,the photosynthesis of plants is affected by related factors such as chloroplast development and chlorophyll synthesis.Exploring the functions of related genes in these processes will help us to take advantage of photosynthesis and increase grain yield.In this laboratory,we obtained a rice mutant of the dark green panicle 1,the leaves and panicles of this mutant all showed a dark green phenotype.Meanwhile,they also increased chlorophyll content and enhanced photosynthetic efficiency.This gene has been cloned by map-based cloning.Since DGP1 encodes a protein with unknown function,this experiment needs to select a protein that interacts with it in rice,and the function of this gene is studied through the characteristics of its interaction proteins.A cDNA library on yeast two-hybrid AD(PGADT7)vector was constructed using RNA extracted from various tissues of rice Nipponbare.The DGP1 was constructed on the BD(PGBKT7)vector as a bait protein which was not activated by itself.Yeast two-hybrid method was used to screen the interaction proteins of DGP1.Two chloroplast development-related transcription factors GOLDEN2-LIKE 1(GLK1)and GOLDEN2-LIKE 2(GLK2)were found.The complete GLK1,GLK2 were cloned into the yeast AD vector and the interaction of their encoded protein with DGP1 was verified by one-on-one experiment yeast.The results showed that the yeast that co-transfered AD-GLK1 BD-DGP1 and AD-GLK1,BD-DGP1 all grew normally and appeared blue on the SD/-Leu-Trp-His-Ade+X-@-Gal,indicating that there were two interactions between GLK1,GLK2 and DGP1.The different GCT-box domains of GLK1 and GLK2 were then cloned and the interactions with DGP1 were verified in yeast in the same way.The results showed that the GCT-box domains of GLK1 and GLK2 all interacted with DGP1,and compared with partial GCT-box domain of GLK1 and GLK2,the complete GCT-box domain of them were more likely to interact with DGP1.Fusion proteins GST:DGP1,His:GLK1 and His:GLK2 were obtained using prokaryotic expression,respectively.Since the GST-DGP1 can pull down His:GLK1 and His:GLK2 in the Pull-down experiment,the experimental results further confirm the interaction between GLK1,GLK2,and DGP1.The fusion protein GST:DGP1 abtained by prokaryotic expression and the extracted total rice protein were used as materials to screen other proteins interacting with DGP1 in rice by Pull-down.As a result,the experiments pulled down two protein bands.After mass spectrometry analysis,they were found to be the subunits of RuBisCO,an enzyme involved in photosynthesis.Then the yeast two-hybrid carriers with large and small subunits of RuBisCO were constructed and tested by one-to-one experiment with DGP1 in yeast.As a result,only one small subunit RBCS5 could interact with DGP1,while other subunits did not DGP1 interaction.However,GLK1 and GLK2 screened by yeast two-hybrid in this study were not pulled down in total rice protein.This may be due to the low protein abundance of GLK1 and GLK2 in rice and what is screened out from rice by yeast two-hybrid technique is the structure of their C-terminal part,and the complete interaction between GLK1 and GLK2 and DGP1 is weak.GLK1 and GLK2 are transcription factors associated with chloroplast development,they regulate the development of chloroplasts and the expression of photosynthetic related genes.RuBisCO is a key enzyme in the process of photosynthesis and it is essential for increased photosynthesis and increased photosynthetic efficiency.Therefore,DGP1 may participate in the development of chloroplasts and chlorophyll synthesis through GLK,thereby affecting leaf color and ear color,while interacting with RuBisCO regulates the enzyme activity of RuBisCO and directly affects photosynthesis.However,these studies also need to be confirmed by follow-up experiments.