Preliminary Study On The Function Of GhHHO2 In Nitrogen Metabolism Of Cotton

Nitrogen,as an essential nutrient element for plant growth and development,has a low content in natural soil and is the main limiting factor affecting crop yield.At present,nitrogen fertilizers are widely used in agricultural production activities to increase crop yields,but crops only absorb 30%-50%of the supplied nitrogen fertilizers.Excessive nitrogen application leads to serious pollution of soil and water bodies,which limits the sustainable development of agricultural production in China.Reducing the input of chemical fertilizers has become a key problem that agricultural scientists care about and needs to be solved urgently.Screening and mining high nitrogen utilization genes and cultivating crop varieties with high nitrogen utilization are an effective way to solve the problem.Transcription factors play an important role in the genetic improvement of crop nitrogen use efficiency.As a nitrate-induced transcriptional repressor,HHO2 has been shown to maintain nitrogen and phosphorus balance in plants by coordinating nitrogen and phosphorus uptake in response to changes in soil nutrients by crops.We cloned the GhHHO2 gene from Gossypium hirsutum in the early stage,and obtained the corresponding T₀transgenic plants.In order to explore the physiological function of GhHHO2 and its regulatory mechanism in nitrogen nutrition metabolism,this thesis firstly analyzed the expression characteristics and subcellular localization of this gene in wild-type cotton,and then analyzed its effect on chlorophyll content,nitrogen metabolism-related enzyme activity and nitrogen content were analyzed in T₁transgenic cotton,and their effects on cotton seed yield traits and fiber quality were analyzed.Finally,their target genes were explored.The results are as follows:1.Expression characteristics and subcellular localization of GhHHO2 geneThe expression levels in different tissues and organs of wild-type cotton were detected by real-time quantitative PCR（q RT-PCR）.The results showed that the expression level of GhHHO2 gene was the highest in tender roots,followed by hypocotyls,tender stems,petals,10 DPA,15 DPA and 20 DPA fibers,and the expression levels were relatively low in ovules at each stage.It indicated that the m RNA of GhHHO2 was abundantly enriched in the rapid elongation（5-16 DPA）and secondary wall deposition and thickening stages（16-20 DPA）of cotton fiber development,and was closely related to cotton seed and fiber development.To investigate the function of GhHHO2 gene,the N-terminal fusion expression vectors p LGN-35S-GhHHO2::e GFP and the C-terminal fusion expression vectors p LGN-35S-e GFP::GhHHO2 were transiently expressed in tobacco leaves,and the nuclear dye DAPI was used for co-localization to determine its localization in the nucleus.2.Effects of GhHHO2 gene expression level on chlorophyll content,enzyme activities related to nitrogen metabolism and nitrogen content in cottonThe T₀ generation transgenic cotton seeds obtained were germinated and sown,and the transgenic plants overexpressing GhHHO2（OE-GhHHO2）,gene editing GhHHO2（Cas9-GhHHO2）and RNA interference GhHHO2（RNAi-GhHHO2）of T₁generation were analyzed for physiology and biochemistry.The chlorophyll a,chlorophyll b and carotenoid content of most OE-GhHHO2 transgenic cotton plants increased significantly.The chlorophyll a and chlorophyll b content of most Cas9-GhHHO2 transgenic cotton has increased,and the carotenoid content has increased significantly.The chlorophyll a,chlorophyll b and carotenoid content of RNAi-GhHHO2 transgenic cotton plants were decreased.The results showed that the overexpression of GhHHO2 may inhibit the transport and assimilation of nitrogen,but the homologous functionally redundant genes of this gene or the existence of a negative feedback regulation mechanism lead to the increase of chlorophyll content.The nitrogen metabolism related enzyme activity assays showed that the activities of nitrate reductase（NR）,nitrite reductase（Ni R）,glutamine synthase（GS）and Fd-glutamine synthase（Fd-GOGAT）of OE-GhHHO2 transgenic plants decreased,and the activities of glutaminamidase（GLS）increased compared with the wild type.The activities of NR,GS and Fd-GOGAT were significantly reduced by 14.8%-63.0%,8.0%-24.6%and 13.0%-54.3%.The activities of Ni R,GS,Fd-GOGAT and GLS of Cas9-GhHHO2 transgenic plants increased,and the activities of NR decreased.The activities of Fd-GOGAT and GLS of most plants increased significantly by35.9%-131.8%and 10.7%-36.0%,respectively,while the activity of NR was significantly reduced by 20.7%-54.8%.The activities of Ni R,GS,Fd-GOGAT and GLS of RNAi-GhHHO2 transgenic plants increased,and the activity of NR decreased.The activities of GS and Fd-GOGAT increased by 45.1%-61.1%and 21.8%-35.4%,respectively,while the activity of NR was significantly reduced by 34.1%-48.1%.The measurement of nitrate nitrogen and ammonium nitrogen showed that the accumulation of nitrate nitrogen and ammonium nitrogen in the overexpression,gene editing and RNA interference GhHHO2 transgenic cotton plants were lower than those of the wild type,and the accumulation of nitrate nitrogen and ammonium nitrogen in most plants was reduced by 13.8%-72.4%and 13.1%-54.0%,respectively.In conclusion,overexpression of GhHHO2 will increase the chlorophyll content of cotton,inhibit the activities of NR,Ni R,GS,and Fd-GOGAT under normal nitrogen supply conditions,and increase the activity of GLS,leading to the accumulation of nitrate nitrogen and ammonium nitrogen content reduced in plants.Gene editing and RNA interference of GhHHO2 gene expression will increase the activity of Ni R,GS,Fd-GOGAT,GLS,and inhibit the activity of NR,therefore the plant has little demand for external nitrogen,resulting in the accumulation of nitrate nitrogen and ammonium nitrogen content decreased.3.Effects of GhHHO2 gene on fiber quality and seed yield traits of cottonFiber quality of T₁ generation OE-GhHHO2 and Cas9-GhHHO2 transgenic cotton plants were detected,and the seed yield traits were counted.The results showed that compared with the wild type,the upper half mean length,uniformity index and fiber strength of the transgenic plants increased,while the micronaire value decreased.The upper half mean length of the overexpressed OE-11 line was significantly increased by7.86%-9.55%,and the fiber strength was significantly increased by 17.14%-24.54%.The upper half mean length of the gene-editing Cas9-50-95 line fiber was significantly increased by 16.08%,the fiber strength was significantly increased by 23.30%,and its micronaire value was 4.43,which was significantly decreased by 14.74%.The results showed that overexpression or gene editing of GhHHO2 gene could improve fiber quality,and GhHHO2 gene played an important role in cotton fiber quality.Overexpressing GhHHO2 transgenic plants showed a significant decrease in boll weight,an increase in seed index,and a decrease in lint percentage.The OE-12 line had a very significant increase in seed index and lint index,a very significant decrease in lint percentage,and significant decrease in boll weight;The seed index and lint index of the gene editing lines increased,whereas the lint percentage decreased.In conclusion,compared with wild type,both overexpression and gene editing of GhHHO2 gene can improve cotton fiber quality,increase seed index and lint index,and decrease lint percentage,indicating that GhHHO2 gene plays an important role in the development of cotton seeds and fibers.4.Screening and identification of downstream target genes of GhHHO2 geneThe interaction between GhHHO2 and the nitrogen and phosphorus nutrition metabolism related genes Gh NRT1.1,Gh NRT2.5,Gh PHT1;1,Gh NLP7 and Gh AMT2was detected by dual luciferase assay.The results showed that the expression of GhHHO2 gene driven by promoters of Gh NRT1.1,Gh NRT2.5,Gh PHT1;1,Gh AMT2and Gh NLP7 were significantly inhibited,and the relative activity of luciferase（LUC）was decreased by 210.1%,193.1%,25.1%,75.1%and 395.7%,respectively.The number of core binding sequences GAAT（CA）TTC contained in the promoter fragments of these five genes is 1,3,1,2 and 3,respectively,which indicates that the number of binding sequences and the interaction strength between genes have significant correlation.The results of Electrophoretic Mobility Shift Assay（EMSA）showed that GhHHO2 protein had a direct interaction with the promoter fragments p Gh NRT1.1,p Gh NRT2.5,p Gh NLP7,p Gh AMT1;1,p Gh AMT2,p Gh PHT1;1 and p Gh PHT1;4.This indicated that Gh NRT1.1,Gh NRT2.5,Gh NLP7,Gh AMT1;1,Gh AMT2,Gh PHT1;1 and Gh PHT1;4 are the direct downstream target genes for GhHHO2.