Identification of drought tolerance of transgenic GmXTH1 soybean at germination stage
As can be seen from Table 2, germination potential, germination rate and germination index of soybean seeds were significantly decreased with the increase of PEG concentration, and decreased with the increase of PEG concentration.Under the condition of clear water (CK), the germination potential and germination rate of M18, OEA1, OEA2, IEA1 and IEA2 seeds were the largest and no significant difference, indicating that all strains germinated well under normal conditions.With the increase of PEG concentration, the germinating state of OEA1 and OEA2 transgenic materials with GmXTH1 gene overexpression was significantly stronger than that of control group M18, and the germinating state of materials with GmXTH1 gene interference expression was significantly weaker than that of control group M18.After 5%PEG-6000 treatment, the relative germination potential of OEA1 and OEA2 were significantly higher than that of control group M18, and IEA1 and IEA2 were significantly lower than that of control group M18. Compared with water treatment, the relative germination rate of M18, OEA1 and IEA2 had no significant changes.OEA2 and IEA1 were slightly decreased. The germination index and vigor index of the GmXTH1 overexpressed material OEA1 and OEA2 were extremely significantly higher than that of the control material M18, and the GmXTH1 interfered expression material IEA1 and IEA2 were extremely significantly lower than that of the control material M18.After 10% and 15%PEG-6000 treatment, the relative germination potential, relative germination rate, germination index and vigor index of GmXTH1 gene overexpression material OEA1 and OEA2 were extremely significantly higher than that of control material M18, and the GmXTH1 gene interference expression material IEA1 and IEA2 were extremely significantly lower than that of control material M18.The results showed that the overexpression of GmXTH1 gene could significantly increase the germination potential, germination rate and germination index of soybean seeds under drought stress.
As can be seen from Fig. 1, after 6 days, under the condition of clear water (CK), the root lengths of M18, OEA1, OEA2, IEA1 and IEA2 seeds showed no significant differences, but the number of lateral roots of OEA1 and OEA2 transgenic materials with overexpression of GmXTH1 gene was significantly more than that of IEA1 and IEA2 transgenic materials with interfering expression of GmXTH1 gene.After 5%PEG-6000 treatment, the root length of OEA1 and OEA2 was significantly longer than that of M18, the root length of IEA1 and IEA2 was significantly shorter than that of M18, the root length of OEA1 and OEA2 was 1.7 times of that of IEA1 and IEA2, and the number of lateral roots was also significantly more than that of IEA1 and IEA2.After 10%PEG-6000 treatment, the root length of OEA1 and OEA2 was significantly longer than that of M18, and the root length of IEA1 and IEA2 was significantly shorter than that of M18. The root length of OEA1 and OEA2 was 2.1 times of that of IEA1 and IEA2.After 15%PEG-6000 treatment, the root length of OEA1 and OEA2 was significantly longer than that of M18, and the root length of IEA1 and IEA2 was significantly shorter than that of M18. The root length of OEA1 and OEA2 was 3.1 times of that of IEA1 and IEA2.It can be seen from the phenotype that the overexpression of gene is beneficial to the generation of tested roots and the elongation of taproot during seed germination.
Comparative analysis of plant types of different soybean strains under different drought stress
As can be seen from Fig. 2a, under normal water conditions, OEA1, OEA2, IEA1 and IEA2 showed good phenotypic performance and thick green stalks, which showed no significant difference compared with the control group M18.
As can be seen from Fig. 2b, after seven days of drought treatment, OEA1 and OEA2 lines with overexpression of GmXTH1 had slightly drooping and dark green leaves and strong and upright stalks.IEA1 was expressed by GmXTH1 interference. The plants were moderately wilting, the leaves were moderately drooping, curled and shriveled, and the stalks were bending due to mild drought stress.IEA2 plants with moderate wilting were more serious and drooping, and the stems also showed bending phenomenon. The control material M18 had slightly wilting leaves, slightly drooping leaves, slightly yellowing, and slightly curled and wrinkled edges.After seven days of drought treatment, there were significant differences in the overall phenotypes among different strains.After rehydration for 2h, OEA1 and OEA2, the leaves gradually returned to dark green and the stalks were strong and straight.IEA1 and IEA2 after 24h, the plants gradually stood upright from wilting, and the leaves gradually recovered from drooping to rising dark green.After 12h, M18 control material returned to strong and straight stems with upturned leaves, and there was significant difference in overall recovery.The results showed that the overexpression of GmXTH1 gene was beneficial to the improvement of drought tolerance and recovery of plants, and had a positive effect on the response of plants to drought stress.
As can be seen from Fig. 2c, after 15 days of drought OEA1, the transgenic line with GmXTH1 overexpression, was more severely shrivelled, but a small part of the leaves extended normally and the stalks were relatively erect.OEA2 Plant leaves are seriously wrinkled, but a small part of them are normally extended, and the stalks are relatively erect.In the control group, the leaves of M18 plants were seriously wrinkled and the stems were seriously dehydrated and bent.Transforming GmXTH1 interferes with the expression of IEA1 and IEA2. The leaves of the plants are extremely seriously wrinkled, and the stalks are also dry and short due to extremely severe dehydration.After rehydration, OEA1 and OEA2 gradually returned to the normal growth state of dark green leaves and strong and straight stalks 24h later.IEA1 and IEA2 showed no recovery after rehydration, and the plants dried up and died.The control material M18 plants did not recover after rehydration, and the plants dried up and died.The results showed that the overexpression of GmXTH1 gene was beneficial to the improvement of drought tolerance and recovery of plants, and had a positive effect on the response of plants to drought stress.
Comparative analysis of root systems of different soybean strains under different drought stress
As can be seen from Table 3, under normal water conditions, the total root length, surface area, total root projection area, root volume, mean root diameter, total cross number of roots and total root tip number of OEA1 transplants over expressed with GmXTH1 were significantly higher than those of control material M18.The total root length, root volume, average root diameter, total cross number and total root tip number of OEA2 transgenic line with GmXTH1 over expression were significantly higher than those of control group M18, and its root surface area and total projected area were significantly higher than those of control group M18. The total root length and mean root diameter of IEA1 transgenic lines were significantly lower than that of control group M18, and the root volume was significantly lower than that of control group M18. The root surface area, total projection area, total cross number and total root tip number showed no difference with that of control group M18.The total root length, total root projection area, mean root diameter, total root crossover number and total root tip number of IEA2 transgenic lines with GmXTH1 over expression were significantly lower than those of the control material M18, and the root surface area and volume were significantly lower than those of the control material M18.
As can be seen from Table 3, in the case of 7 days of drought, the total root length, surface area, total root projection area, root volume, total cross number and total root tip number of OEA1 transgenic lines over expressed with GmXTH1 were significantly higher than those of control group M18, and the average root diameter showed no difference with that of control group M18.The total length, surface area, total projection area, root volume and total cross number of roots of OEA2 transgenic lines over expressed with GmXTH1 were significantly higher than those of control group M18, and there was no difference between the total projection area, average diameter and total number of root tips of OEA2 transgenic lines overexpressed with GmXTH1 and control group M18.The total root length, surface area, total root projection area, root volume, total cross number and total root tip number of IEA1 transgenic lines were significantly lower than the control material M18, and the mean root diameter was significantly lower than the control material M18.The total root length, surface area, total root projection area, root volume, total root crossover number and total root tip number of IEA2 transgenic lines were significantly lower than the control material M18, and the average root diameter was significantly lower than the control material M18.
As can be seen from Table 3, the total root length, surface area, total root projection area, root volume, mean root diameter, total cross number and total root tip number of OEA1 transgenic lines over expressed by GmXTH1 were significantly higher than those of control group M18 under the condition of drought for 15 days.The total root length and total root tip number of OEA2 transgenic lines over expressed with GmXTH1 were significantly higher than those of the control material M18, and the root surface area was significantly higher than that of the control material M18. There were no differences in the total root projection area, root volume, average root diameter, and total cross number of roots of the control material M18.The total root length, total cross number and total root tip number of IEA1 transgenic lines were significantly lower than those of the control material M18, and the average diameter surface area, total root projection area and root volume of IEA1 transgenic lines were not different from those of the control material M18.The total root length, surface area, total root projection area, root volume, total cross number and total root tip number of IEA2 transgenic lines were significantly lower than those of control group M18, and the mean root diameter had no difference with that of control group M18.
As can be seen from Table 3, the total root length, surface area, total root projection area, root volume, mean root diameter, total cross number and total root tip number of all strains increased significantly after 7 days of drought compared with normal conditions, but OEA1 and OEA2 strains over expressed by GmXTH1 were more significant.The total root length, surface area, total root projection area, root volume, mean diameter, total cross number and total root tip number of each strain decreased significantly after 15 days of drought compared with 7 days of drought, but the decrease amplitude of OEA1 and OEA2 in GmXTH1 over expression lines was small.
The above results indicated that the overexpression of GmXTH1 gene could significantly increase the total root length, surface area, total projection area, root volume, mean root diameter, total cross number and total root tip number of the plant root system, which promoted the more developed root system and was more conducive to the absorption of water and minerals.
Physiological and biochemical analysis of GmXTH1 transgenic soybean at seedling stage
As can be seen from Fig. 3a, without drought treatment, RWC of leaves of different soybean strains had significant differences as a whole.After 7 days of drought treatment, the RWC of OEA1 and OEA2 leaves was significantly higher than that of M18 control group 79.14%, 80.56% and 83.97%, respectively, and the RWC of IEA1 and IEA2 leaves was significantly lower than that of M18 control group 79.14%, 75.68% and 76.25%, respectively.There were significant differences in RWC among leaves after 7 days of drought.After 15 days of drought treatment, the RWC of OEA2 and OEA1 leaves was significantly higher than that of M18 in the control group 66.93%, 79.24% and 78.54%, respectively, and the RWC of IEA2 and IEA1 leaves was significantly lower than that of M18 in the control group 66.93%, 60.35% and 61.77%, respectively.The RWC of leaves was significantly different after 15 days of drought.The results indicated that the overexpression of GmXTH1 gene could significantly delay the decrease of RWC and reduce transpiration of water in leaves.
As can be seen from Fig. 3b, MDA content of different strains showed no significant difference without drought treatment.After 7 days of drought treatment, the MDA content of OEA2 and OEA1 was significantly lower than that of M18, and the MDA content of IEA2 and IEA1 was significantly higher than that of M18, and the growth rates of MDA content of OEA2 and OEA1 were 46.57% and 37.50%, respectively, significantly lower than that of control M18 73.67%.The MDA content of IEA2 and IEA1 increased by 200.24% and 206.95%, respectively.After 15 days of drought treatment, the MDA content of OEA2 and OEA1 was significantly lower than that of M18, the MDA content of IEA2 and IEA1 was significantly lower than that of M18, and the MDA content of OEA2 and OEA1 increased by 104.30% and 82.94%, respectively, significantly lower than that of control M18 144.84%.The MDA content of IEA2 and IEA1 increased by 297.72% and 296.99%, respectively.The results indicated that the overexpression of GmXTH1 gene could slow down the peroxidation degree of membrane lipid.
As can be seen from Fig. 3c, there was no significant difference in POD activity between different strains before drought treatment (0d).After 7 days of drought treatment, the POD activity of OEA2 and OEA1 was significantly higher than that of M18, and the POD activity of IEA2 and IEA1 was significantly lower than that of M18, and the growth rates of POD activity of OEA2 and OEA1 were 176.45% and 155.24% respectively, which were significantly higher than that of control M18 113.54%.The POD activity of IEA2 and IEA1 increased by 67.66% and 61.27%, respectively.After 15 days of drought treatment, the POD activity of OEA2 and OEA1 was significantly higher than that of M18, and the POD activity of IEA2 and IEA1 was significantly lower than that of M18, and the growth rates of POD activity of OEA2 and OEA1 were 69.12% and 65.59% respectively, which were significantly higher than that of 57.00% of control M18.The POD activity of IEA2 and IEA1 increased by 44.65% and 40.94%, respectively. The results indicated that the effective removal of harmful substances during the seedling stage of soybean transgenic GmXTH1 gene overexpression could produce more protective enzymes and resist the damage caused by drought.
As can be seen from Fig. 3d, without drought treatment, SOD activity of different strains showed no significant difference.After 7 days of drought treatment, the SOD activities of OEA2 and OEA1 were significantly higher than that of M18, and the SOD activities of IEA2 and IEA1 were significantly lower than that of M18, and the SOD activity growth rates of OEA2 and OEA1 were 126.89% and 156.57%, respectively, significantly higher than that of control M18 94.65%.The SOD activity of IEA2 and IEA1 increased by 84.98% and 74.63%, respectively.After 15 days of drought treatment, the SOD activities of OEA2 and OEA1 were significantly higher than that of M18, and the SOD activities of IEA2 and IEA1 were significantly lower than that of M18, and the SOD activity growth rates of OEA2 and OEA1 were 56.63% and 59.50% respectively, which were significantly higher than that of control M18 33.55%.The SOD activity of IEA2 and IEA1 increased by 21.88% and 22.88%, respectively.The results indicated that the effective removal of harmful substances during the seedling stage of soybean transgenic GmXTH1 gene overexpression could produce more protective enzymes and resist the damage caused by drought.
Relative expression levels of target gene and other endogenous genes in transgenic GmXTH1 soybean at seedling stage
As can be seen from Fig. 4a, under normal water conditions, the expression level of OEA1 GmXTH1 in soybean roots and leaves was increased by 44.39% and 50.00% respectively.The expression level of OEA2 strain GmXTH1 in soybean roots and leaves was increased by 56.37% and 42.90% respectively.The expression level of GmXTH1 of IEA1 strain was decreased by 74.74% in soybean root and 61.31% in leaf.The expression level of GmXTH1 of IEA1 strain was decreased by 44.71% in soybean root and 85.39% in leaf.
As can be seen from Fig. 4b, after 7 days of drought, the expression level of OEA1 GmXTH1 in soybean roots and leaves increased by 113.61%.The expression level of OEA2 strain GmXTH1 in soybean roots and leaves was increased by 120.38% and 171.32% respectively.The expression of IEA1 strain GmXTH1 in soybean roots and leaves was reduced by 32.40% and 68.00% respectively.The expression level of GmXTH1 of IEA1 strain was decreased by 60.50% in soybean roots and 34.48% in soybean leaves.
According to Fig. 4c, under normal water conditions, the expression level of OEA1 strain JCVI-FLGM-14H24 in soybean roots and leaves was increased by 50.52% and 7.92% respectively.The expression level of OEA2 strain JCVI-FLGM-14H24 in soybean roots and leaves was increased by 43.40% and 14.87% respectively.The expression level of IEA1 strain JCVI-FLGM-14H24 was decreased by 33.10% in soybean root and 61.31% in soybean leaf.The expression level of IEA1 strain JCVI-FLGM-14H24 was decreased by 49.65% in soybean roots and 74.30% in soybean leaves.The above results indicated that the overexpression of GmXTH1 promoted the expression of JCVI-FLGM-14H24 under normal water conditions, while the interference of GmXTH1 inhibited the expression of JCVI-FLGM-14H24.
As can be seen from Fig. 4d, after 7 days of drought, the expression level of OEA1 strain JCVI-FLGM-14H24 in soybean roots and leaves increased by 80.25% and 12.51%.The expression level of OEA2 strain JCVI-FLGM-14H24 was increased by 48.45% in soybean roots and 10.96% in leaves.The expression level of IEA1 strain JCVI-FLGM-14H24 was decreased by 19.34% in soybean roots and 12.95% in soybean leaves.The expression level of IEA1 strain JCVI-FLGM-14H24 was decreased by 25.26% in soybean roots and 13.55% in soybean leaves.The above results indicated that the overexpression of GmXTH1 promoted the expression of JCVI-FLGM-14H24, while the interference expression of GmXTH1 inhibited the expression of JCVI-FLGM-14H24 under drought for 7 days.
As can be seen from Fig. 4e, under normal water conditions, the expression level of OEA1 strain GmWRKY35 in soybean roots and leaves increased by 80.25% and 536.43%.The expression level of OEA2 strain GmWRKY35 was decreased by 78.61% in soybean roots and increased by 748.55% in leaves.The expression level of IEA1 strain GmWRKY35 in soybean roots and leaves was increased by 471.60% and 1608.90% respectively.The expression level of IEA2 strain GmWRKY35 in soybean roots and leaves was increased by 56.37% and 1656.95% respectively.The above results indicated that the low expression level of GmXTH1 under normal water condition was conducive to the expression of GmWRKY35.
As can be seen from Fig. 4f, after 7 days of drought, the expression of OEA1 strain GmWRKY35 in soybean roots decreased by 49.48% and increased by 44.89% in leaves.The expression level of OEA2 strain GmWRKY35 was decreased by 30.27% in soybean roots and increased by 39.47% in leaves.The expression level of IEA1 strain GmWRKY35 was decreased by 56.02% in soybean roots and increased by 25.70% in soybean leaves.The expression level of IEA1 strain GmWRKY35 was decreased by 69.75% in soybean roots and 11.12% in soybean leaves.The above results indicated that the overexpression of the target gene GmXTH1 was beneficial to the expression of GmWRKY35 in soybean leaves under drought for 7 days, while the expression of the target gene GmXTH1 inhibited the expression of GmWRKY35 in soybean roots, and the higher the expression level, the weaker the inhibition.
As can be seen from Fig. 4g, under normal water conditions, the expression level of OEA1 strain GmPLR-2 in soybean roots and leaves was increased by 217.11% and 8171.06% respectively.The expression level of OEA2 strain GmPLR-2 was decreased by 95.00% in soybean roots and increased by 3512.69% in leaves.The expression level of GmPLR-2 of IEA1 strain was decreased by 44.91% in soybean root and increased by 6344.52% in soybean leaf.The expression level of GmPLR-2 of IEA1 strain was decreased by 85.39% in soybean roots and increased by 6711.97% in soybean leaves.The results showed that the expression of target gene GmXTH1 inhibited the expression of GmPLR-2 in soybean roots and promoted the expression of GmPLR-2 in soybean leaves under normal water conditions.
According to Fig. 4h, after 7 days of drought, the expression level of OEA1 strain GmPLR-2 in soybean roots and leaves decreased by 25.26% and 19.34%.The expression level of OEA2 strain GmPLR-2 in soybean roots was increased by 5.70% and decreased by 15.03% in leaves.The expression level of GmPLR-2 of IEA1 strain was decreased by 22.62% in soybean roots and increased by 227.16% in soybean leaves.The expression of IEA1 strain GmPLR-2 was decreased by 60.09% in soybean roots and increased by 244.62% in soybean leaves.The above results indicated that the target gene GmXTH1 was overexpressed and inhibited the expression of GmPLR-2 in soybean leaves under drought for 7 days, while the target gene GmXTH1 interfered with the expression and promoted the expression of GmPLR-2 in soybean leaves.
Fig. 4. Relative expression levels of GmXTH1 and JCVI-FLGM-14H24 in different transgenic soybean lines under different drought conditions at seedling stage (a.Under normal circumstances, the relative expression of GmXTH1 in OEA1,OEA2, IEA1 and IEA2 roots and leaves;b. Relative expression levels of GmXTH1 in OEA1,OEA2, IEA1 and IEA2 roots and leaves after 7 days of drought :c.Under normal conditions, the relative expression levels of JCVI-FLGM-14H24 in OEA1,OEA2, IEA1, and IEA2 roots and leaves: d. JCVI-FLGM-14H24 in OEA1,OEA2, IEA1, and IEA2 roots and leaves:e.Relative expression levels of GmWRKY35 in roots and leaves of OEA1,OEA2, IEA1 and IEA2;f. The relative expression levels of GmWRKY35 in OEA1,OEA2, IEA1 and IEA2 roots and leaves;g. The relative expression levels of GmPLR-2 in OEA1,OEA2, IEA1 and IEA2 roots and leaves;h. The relative expression levels of GmPLR-2 in OEA1,OEA2, IEA1 and IEA2 roots and leaves)