Identification Of TCP And NHX Gene Families In Amaranth And Their Functions In Response To Salt Stress

Amaranthus tricolor L.is an annual dicotyledonous herb of the genus Amaranthus in the order Amaranthaceae,with rich nutritional value,wide cultivation area and long history of cultivation,which has received wide attention in recent years.Nowadays,soil salinity is one of the major abiotic stresses that limit plant growth and development and has been a global research hotspot.Therefore,it is especially important to investigate the salt tolerance mechanism of plants,to explore the key salt tolerance genes,and to enhance the salt tolerance ability of plants.TCP genes mainly regulate the growth and development of plant flowers,buds and leaves,and respond to salt stress.The NHX gene mediates Na⁺/H⁺exchange,maintains the ion balance inside and outside the cell,and reduces the toxic effects caused by Na⁺accumulation.Since the physiological and molecular mechanisms of Amaranthus response to salt stress are still unclear.In this study,the effects of different concentrations of Na Cl solution on the nutritional growth and main nutrients,photosynthetic characteristics and physiological and biochemical indexes of amaranth were investigated using’Suxian NO.1’as the test material;And the response of AtrTCP and AtrNHX genes to salt stress was examined using q RT-PCR,and the functions of AtrTCP1 and AtrNHX11were verified by transformation of Arabidopsis thaliana.The test results are as follows:1.Effects of salt stress on the growth and nutrients of AmaranthusAmaranthus seedlings at the 3-leaf stage were treated with different concentrations of salt stress(0 mmol·L^-1,50 mmol·L^-1,100 mmol·L^-1and200 mmol·L^-1Na Cl).The results showed that the height of amaranth plants under 50 mmol·L^-1Na Cl treatment was higher than the rest of Na Cl treatment,and the root elongation and the number of fibrous roots increased;the amaranth plants under 200 mmol·L^-1Na Cl treatment were short and the root elongation and the number of fibrous roots increased were inhibited.The study on the main nutrients of amaranth revealed that the highest flavonoid content of amaranth plants was found under 50mmol·L^-1Na Cl treatment and the lowest under 200 mmol·L^-1Na Cl treatment.The differences in flavonoid content were more significant under different concentrations of Na Cl treatment as the time was increased.In contrast,the trend of beet pigment content was opposite to that of flavonoids,with the highest beet pigment content under 200mmol·L^-1Na Cl treatment.2.Effects of salt stress on photosynthetic characteristics and physiological and biochemical indexes of AmaranthusThe determination of chlorophyll content of amaranth leaves under salt stress treatment revealed that the total chlorophyll content showed a trend of increasing and then decreasing,with the highest total chlorophyll content in amaranth leaves under 100 mmol·L^-1Na Cl treatment and the lowest total chlorophyll content in amaranth leaves under 200 mmol·L^-1Na Cl treatment.Analysis of chlorophyll fluorescence of amaranth leaves under salt stress treatment revealed that chlorophyll fluorescence parameters were down-regulated with increasing salt solution concentration.The determination of root vigor revealed that the root vigor of amaranth plants under 50 mmol·L^-1Na Cl treatment was the strongest.The physiological and biochemical indexes were measured and found that peroxidase activity and malondialdehyde content increased significantly with increasing Na Cl concentration,reaching a maximum under 200mmol·L^-1Na Cl treatment;Catalase activity was highest under 100mmol·L^-1Na Cl treatment.3.Identification of AtrTCP gene family in amaranth and gene expression profile under salt stressThe AtrTCP gene family has 14 members,all localized in the nucleus;The AtrTCP protein length is in the 230-721 aa interval,with a predicted relative molecular weight of 25.25-78.57 k D and an isoelectric point of 6.15-9.45,and contains 15 conserved motifs;All were unstable proteins except AtrTCP11,which was a stable protein.mi RNA predicted two AtrTCP family members,AtrTCP2 and AtrTCP6,as mi R319 target genes.The q RT-PCR results showed that the gene family had different expression patterns under different concentrations of salt stress treatment;among them,AtrTCP1 had the lowest expression under 50 mmol·L^-1Na Cl treatment and the highest expression under 200 mmol·L^-1NaCl treatment.q RT-PCR analysis in various parts of amaranth revealed that AtrTCP3 and AtrTCP12 were highly expressed in the leaves,and the rest of the family members were all expressed in higher amounts in the roots.4.Identification of AtrNHX gene family in amaranth and gene expression profile under salt stressBased on amaranth transcriptome data,12 AtrNHX family member genes were screened,and the molecular weight of this family member ranged from 15.45-61.04 k D;The isoelectric point ranged from 5.30-9.28;All members of this gene family were non-hydrophilic proteins.Subcellular localization revealed that 12 genes were localized to the vesicles.AtrNHX7 and At NHX3 were more closely related,and AtrNHX2and AtrNHX11 were more closely related to At NHX4.The expression pattern of NHX family members in amaranth treated with different concentrations of salt solution was analyzed by q RT-PCR.AtrNHX11showed the highest expression under 100 mmol·L^-1NaCl treatment,while the rest of the members were all expressed at 50 mmol·L^-1Na Cl treatment.q RT-PCR analysis in various parts of amaranth revealed that most family members were highly expressed in roots or stems.5.Functional validation of AtrTCP1 and AtrNHX11 genes in AmaranthusSeed germination tests were conducted on wild-type and transgenic Arabidopsis thaliana T2 generation seeds,and it was found that the seeds of transformed plants germinated earlier under non-salt stress treatment,and the germination rate and germination potential were higher than those of wild-type.At 14 days of seed germination,the root length of35S::AtrTCP1 transformed plants under non-salt stress treatment was not significantly different from that of the wild type;The root length of35S::AtrNHX11 transformed plants was smaller than that of the wild type.The root lengths of the wild-type,35S::AtrTCP1 and 35S::AtrNHX11transformed plants treated with salt stress were smaller than those of the non-salt stress treatment.When Arabidopsis grew to 6 leaves,wild-type and Arabidopsis transgenic plants were treated with 150 mmol·L^-1Na Cl,and all treated plants were shorter and had smaller leaf size and number of leaves than non-salt stress treatments.Under salt stress,35S::AtrNHX11 transformed plants flowered at8.8th day post-treatment when they grew to 10.2 leaves;35S::AtrTCP1transformed plants flowered at 16.2th day post-treatment when they grew to 21.8 leaves;And the wild type flowered at 18.4th day when it grew to21.6 leaves.Under non-salt stress treatment,35S::AtrNHX11 transformed plants flowered when they grew to 9.8 leaves at the 9.2th day of continued culture;35S::AtrTCP1 transformed plants flowered when they grew to 16.2 leaves at the 13.2th day of continued culture;And the wild type flowered when they grew to 16.6 leaves at the 13.8th day of continued culture.The chlorophyll content of wild-type Arabidopsis thaliana treated with salt stress was significantly lower than that of non-salt stress treatment;while the chlorophyll content of 35S::AtrTCP1 and35S::AtrNHX11 transformed plants was significantly higher than that under non-salt stress treatment.The chlorophyll content of 35S::AtrTCP1and 35S::AtrNHX11 transformed plants was higher than that of wild type under both salt stress and non-salt stress.Analysis of salt stress on the expression of related genes in Arabidopsis transformed plants showed that the expression of GUS genes in 35S::AtrTCP1 and 35S::AtrNHX11 transformed plants was significantly higher under salt stress than under non-salt stress;The expression of the target gene was significantly higher in wild-type,35S::AtrTCP1 and 35S::AtrNHX11 transformed plants under salt stress than under non-salt stress.Analysis of transgenic correlations under salt stress revealed that AtrTCP1 showed a highly significant positive correlation with salt concentration and AtrNHX11 showed a significant positive correlation with salt concentration;AtrTCP1 and AtrNHX11showed a significant positive correlation.