| Eucommia ulmoides is a unique economic tree with multiple functions in China.Leaves are an important raw material for the sustainable utilisation of E.ulmoides.It is useful for the development and utilisation of E.ulmoides to study genes related to the growth and development of leaves.The auxin response factor(ARF)is an important transcription factor that plays a role in the regulation of growth and development of leaves.The genome sequencing data of E.ulmoides can be used to study the ARF gene family and screen the ARF genes regulating leaf growth.This will fill the gaps in ARF gene studies,provide a reference for other gene research,and has great significance for the exploitation and utilisation of E.ulmoides.In this study,the ARF gene family in E.ulmoides was identified in the genome data.Combined with the data of RNA-seq and small RNA sequencing,the function of the ARF gene family in E.ulmoides was predicted by expression analysis.EuARF19.2,a key gene significantly affecting the growth and development of leaves,was selected.The EuARF19.2 plant expression vector was constructed and transformed into Arabidopsis thaliana to verify its function.Finally,a callus regeneration system for E.ulmoides leaf explants was optimised,and an Agrobacterium-mediated transformation system of leaf callus was established to carry out the genetic transformation of EuARF19.2.The main results were as follows:(1)A total of 18 EuARFs were identified in the E.ulmoides genome and were divided into five classes.EuARFs in the same class had similar physiochemical characteristics,protein structure,and may perform similar functions.The results of expression profiling and q RT-PCR reflected that EuARFs were expressed in organs(roots,stems,and leaves)at different developmental stages(young and mature)under normal growth conditions.The expression of EuARF19.2 in leaves was significantly higher than other EuARFs.It is speculated that EuARF19.2 plays an important role in regulating the growth of leaves.(2)Analysis of the correlation between EuARF19.2 expression and leaf area showed that the expression of EuARF19.2 at the early leaf development stage was positively correlated with leaf area.It is suggested that EuARF19.2 can regulate the development of leaf morphology and promote the increase of leaf area.The expression of EuARF19.2 in stem leaves under IAA treatments showed that the expression of EuARF19.2 was significantly increased with the treatment of exogenous IAA.It is suggested that EuARF19.2 could respond to exogenous IAA.(3)The full-length cDNA sequence of EuARF19.2 was obtained and the pBI121-EuARF19.2 expression vector was synthesised.Agrobacterium-mediated transformation of Arabidopsis thaliana was carried out.T3 transgenic plants were obtained by screened with Kanamycin(Kan)and confirmed by PCR.Phenotype observation of T3generation positive transgenic plants showed that EuARF19.2 promoted the increase of leaf area.(4)The callus regeneration system for E.ulmoides leaf explants was optimised.The optimum medium for callus induction was MS+8.1?mol·L-1 NAA+4.4?mol·L-1 6-BA,with an induction rate of 100%.The optimum medium for callus proliferation was MS+0.27?mol·L-1 NAA+6.7?mol·L-1 6-BA,with a multiplication coefficient of 304±36%,browning rate of 16±4%,and subculture period of 18-20 days.The optimum medium for the induction of adventitious buds in callus was?MS+0.27?mol·L-1 NAA+4.4?mol·L-1 6-BA,with an induction rate of 83±10.0%.The optimal medium for adventitious bud rejuvenation was?MS+0.054?mol·L-1 NAA+4.4?mol·L-1 6-BA,with an average growth length of 2.47±1.33 cm.(5)Using the GUS transient expression rate as the standard,the transformation factors affecting Agrobacterium-mediated transformation of E.ulmoides leaf callus were evaluated.The optimal combination of transformation factors was obtained as follows:5 days of pre-culture,10 min of infection time,3 days of co-cultivation,and a selection medium of?MS+0.054?mol·L-1 NAA+4.4?mol·L-1 6-BA+200 mg·L-1Cef+70 mg·L-1Kan.Using this system,4 resistant buds were obtained by genetic transformation of the pBI121-EuARF19.2 expression vector.PCR analysis and GUS histochemical staining indicated that target gene had been integrated into the genome of these resistant buds.Phenotype observations and expression analysis of EuARF19.2 proved that overexpression of EuARF19.2 can increase the leaf area. |
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