Functional Analysis Of DELLA1,GID1,GA2ox1 Gene In The Regreening Process Of Pear Iron Deficiency Chlorosis

China is the country with the largest pear cultivation area in the world.In 2017,the pear cultivation area in China was about 960,000 hm²and the total output was about 16.53million tons,accounting for 69.1%and 68.4%of the world’s total area and total output respectively.Communist-held areas of the Yellow River is one of pear producing in our country,where‘Dang Shansuli pear’was widely cultivated.In recent years,a variety of pears such as‘Huangjin pear’,‘Yuanhuang pear’,‘Qiuyue pear’,‘Cuiguan pear’and other sand pear varieties were introduced.Iron is an essential trace element in the growth and development of pear trees and is involved in many metabolic reactions including photosynthesis.Iron deficiency will lead to the inhibition of photosynthetic electron transfer,photosynthesis and chloroplast synthesis,and the young leaves will exhibit the typical iron deficiency and chlorosis,which will seriously affect the development of pear industry.Due to the soil characteristics of the Yellow River’s Communist-held areas,the effective iron absorption of the root system was blocked,resulting in different degrees of iron deficiency chlorosis.Which make a vital sense to the development of the regional pear industry.Preliminary results showed that spraying 200 mg·L^-1exogenous GA₃could partlyalleviate the symptoms of young leaf yellowing and make them regreen.Based on this,normal leaf（NL）and chlorosis leaf（CL）of pear were used as test materials in this experiment,we analyzed the different expression of GID1.3,DELLA1.4 and GA2ox1involved in gibberellin transduction pathway by using the Quantitative real-time PCR.We had preliminary explored the application of virus induced gene silencing（VIGS）technology in the pear leaf and Nicotiana benthamiana.By establishing super fusion vectors,the effects of GID1.3,DELLA1.4,GA2ox1 in the regreening progress of pear iron-deficiency chlorosis and NL yellowing were analyzed in two ways.Moreover,Chlorophyll content and the ferrous iron contents in each sample were further determined by the application of spectrophotometer and piero pyryluim chromogenic method.Finally,we analyzed the tolerance of over-expression model plant-Arabidopsis thaliana to iron deficiency.The functions of DELLA1.4,GID1.3 and GA2ox1 in the process of leaf regreening were primarily identified.The main research results were showed as follows:1.The full-length of GID1.3 is 1041 bp,including theα/βcatalytic domain-abhydrolase-3 and coesterase.GA2ox1 gene has a total length of 1014 bp and contains a ferrous-glutarate dioxygenase domain-Fe II-20G＿Oxy while DELLA1.4 reaches a length of1632 bp,which only contains GRAS domain,and may be involved in the protein interaction of important transcription factor PIF3/4 related to plant photomorphogenesis;The protoplast results of agrobacteria-mediated transformation showed that GA2ox1 and DELLA1.4 were located in the cytoplasm while GID1.3 was located in the nucleus;2.DELLA1.1,DELLA1.2 and DELLA1.4 in CL were significantly lower than NL,while the expression levels of DELLA1.3 in CL were significantly higher than NL.The expression of GID1.1,GID1.2,GID1.3 and GA2ox1 in CL were significantly higher than CL.Under iron deficiency stress,the content of ferrous iron in CL was significantly lower compared to NL and exogenous GA₃application could significantly increased the content of ferrous iron in regreening leaf（RL）;Exogenous GA₃treatment significantly increased the expression of GID1.3 compared to NL while significantly decreased the expression of DELLA1.4.Otherwise,The expression of GA2ox1 in RL was significantly increased compared to NL but significantly decreased compared to CL;On the other hand,the expression of metal transporter NRAMP1.2 in CL were significantly lower than NL,there were no differences of NAP1,NAP1.2,REM5,REM5.2,NAP1 and NAP1.2 between NL and CL,but in RL,NAP1、NAP1.2 were significantly increased.Respectively,REM5 and REM5.2 were significantly up-regulated in RL.However,the expression of NAP2 in RL was significantly reduced;3.The over-expression vectors GID1.3-1300,DELLA1.4-1300,GA2ox1-1300 and the silent vectors GID1.3-TRV1,DELLA1.4-TRV1,GA2ox1-TRV1 were constructed,which were injected to the leaf of‘Qiuyue pear’.The instantaneous expression results showed that the injection area of the pear CL after injected by GID1.3-1300,GA2ox1-1300and DELLA1.4-TRV1 came out to the phenomenon of regreening,while the NL injected with DELLA1.4-1300,GID1.3-TRV1,and GA2ox1-TRV1 presented chlorosis in the injection area.Meanwhile,no significant changes in leaf were observed after application of1300 and TRV2 control;The determination of chlorophyll content in injection area showed that the chlorophyll content in RL significantly increased compared to CL,but was significantly lower than NL.The chlorophyll content in the chlorosis area of NL decreased significantly,but was significantly higher than that in CL;The ferrous iron content in the injection aera of each treatment showed that compared with NL and CL,the content in the control groups showed no changes,while it significantly increased in the injection aera of GID1.3-1300,GA2ox1-1300 and DELLA1.4-TRV1 treatment.In addition to GA2ox1-TRV1,the ferrous iron content in the leaf treated with DELLA1.4-1300 and GID1.3-TRV1 was significantly lower than NL but higher than CL;The phenomenon of Nicotiana benthamiana showed that the same with instantaneous in pear leaf.And in addition,the ferrous iron content of GID1.3-TRV1 and GA2ox1-TRV1was remarkably decreased than the control,there was no significant change between H₂O and TRV2-TRV1.However,the ferrous iron content in the injection aera of DELLA1.4-TRV1 was significantly higher than control.4.The root system of GA2ox1 transgenic arabidopsis was short and dense,and the root system of GID1.3 was significantly elongated,but there was no significant differences between the root system of DELLA1.4 and that of WT transgenic lines;The seed germination rate of GA2ox1 transgenic arabidopsis was significantly inhibited while it of GID1.3 and DELLA1.4 showed no significant differences to WT.After two true leaves grown,transplanting them in vegetative soil,the results showed that the blot state and flowering period of GA2ox1 transgenic lines delayed about 3 days than other lines while others showed no remarkably differences.The leaf shape of GID1.3 was long and peak,while the leaf area of GA2ox1 was small but round.5.Transgenic arabidopsis cultured in iron-deficient medium of 25mmol·L^-1(Fe²⁺)showed that GA2ox1 over-expression lines’leaf kept green while GID1.3,DELLA1.4 and WT turned into chlorosis.