| Phospholipase D (PLD) can hydrolyze phospholipid to produce phosphatidic acid (PA). PA is very important in plant development and stress. PLD is found in many species including, Arabidopsis (12 members), rice (17 members), poplar (18 members), grape (11 members) and so on. The members of PLD in soybean(GmPLD) have been not identified and the function of GmPLD in stress has not investigated. The members of GmPLD have identified in this article and the functions of GmPLD have been preliminarily investigated. GmPLDy, one of the GmPLD, have been cloned and transformed into Arabidopsis to investigate the function in salt stress.18 members of PLD have been found in soybean through genomic analysis. GmPLD have been divided into six types including a (3), β (4), γ, δ (5), ε (2) and ζ(3), based on the protein domain, sequence similarity and evolutionary relationship. The first five types contain C2 domain, called C2-PLD and the last type contain PX/PH domain, called PX/PH-PLD. qRT-PCR was used to detect the expression of GmPLD in various tissues. GmPLDal, GmPLDa2 and GmPLDβ2 were highly expressed in most of tissues. GmPLDδ5 was only expressed in flowers. GmPLDζ1 was predominantly expressed in flowers and early pods. GmPLDβ1、P4 and e2 were expressed much lower in all of the tissues. To investigate the effect of salt and ABA stress to GmPLD. The soybean seedlings were treated with 250mM NaCl and 100μM ABA. In the salt stress, GmPLDal and α2 were increased during the 8 hours, however, GmPLDα3, δ3, δ4 increased before 4 hours and decreased to the base level after 8 hours. Because some of GmPLD were drastic changes in the control of ABA treatment, the effect of ABA treatment to GmPLD were not clear. PLD activity of GmPLDa has been detected. The results showed that GmPLDα 1 had high PLD activity towards PC (phosphatidylcholine), PE (phosphatidyl ethanolamine), PG (phosphatidyl glycerol) and PS (phosphatidylserine). GmPLDa2 showed no activity in the buffer and substrates. GmPLDa3 was detected low PLD activity towards to PC.There is only one member in GmPLDy. The soybean seedlings were treated with 250mM NaCl. In roots, the expression level of GmPLDy increased at 0.5h aftertreatment and returned to normal level at 1h after treatment. In leaves, the expression level of GmPLDy showed a sharp rise at 1h after treatment and returned to normal level at 4h after treatment. The results proved GmPLDy may take part in salt stress. The promoter of GmPLDy has been analyzed and two elements of "TGGGCC" have been found. GmPLDy was localized on mitochondria. To confirm the function in salt stress, GmPLDy was cloned and transformed into Arabidopsis to obtain the transgenic plants. The transgenic plants and WT were treated with 200mM NaCl and the germination of transgenic seeds increased. The results showed that GmPLDy is important in seed germination. GmPLDy transgenic seeds increased the oil content.Soybean is the important commercial crop and its product of oil is the large part of edible oil, especially in China. So increasing the oil content can enhance the economic benefits. The most important composition of soybean oil is TAG which was synthesized through the Kennedy pathway. GPAT (Glycerol-3-phosphate acyltransferase), LPAT (lysophosphatidic acid acyltransferase) and DGAT (Acyl-CoA:Diacylglycerol acyltransferase) are the most important enzyme in this pathway. So LPAT and DGAT in soybean were studied to increase the oil content.8 members of membrane-bound DGAT have been identified in soybean and named as GmDGATIA, GmDGATIB, GmDGAT1C, GmDGAT2A, GmDGAT2B, GmDGAT2C, GmDGAT2D and GmDGAT2E based on the evolutionary relationship. The DGAT activity results showed that GmDGATIA, GmDGATIB, GmDGAT2A, GmDGAT2B have the activity, however, GmDGAT2D showed no activity in the detected substrates and buffer. GmDGATIA, GmDGATIB had higher activity than GmDGAT2A, GmDGAT2B. GmDGATIA, GmDGATIB, GmDGAT2A, GmDGAT2B, GmDGAT2D were cloned and transformed into Arabidopsis. The oil was extracted from the transgenic plants seeds and the results showed that GmDGATIA, GmDGATIB transgenic seeds increase the oil content in a range of 15.4%-21.7%. GmDGAT2D transgenic seeds increased oil content but they were showed no significant difference compared with WT and vector transgenic seeds. The two lines of GmDGATIA transgenic plants had more rosstte branches compared with vector transgenic plants. The plants grew for 7 weeks and the two lines of GmDGATIA transgenic plants had 5.8 and 6.2 rosstte branches, however, the vector transgenic plants had 2.8 rosstte branches. MAX3 (MORE AXILLARY GROWTH 3) and MAX4, the related genes to branching, expressed lower after bolting in GmDGATIA transgenic plants. GR24, the analogue of strigolactones which were synthesized by MAX, were used to treat GmDGATIA transgenic plants. The results showed that the rosstte branches returned to the normal number compared with the vector transgenic plants.10 LPAT gens were found through genomic analysis in soybean and named as GmLPATIA, GmLPAT2A, GmLPAT2B, GmLPAT2C, GmLPAT2D, GmLPAT3A, GmLPAT4A, GmLPAT4B, GmLPAT5A and GmLPAT5B. All the 10 genes except GmLPAT4A were cloned and transformed into Arabidopsis. GmLPAT2B, GmLPAT2C, GmLPAT2D, GmLPAT4B, GmLPAT5A, GmLPAT5B transgenic plants were obtained. The, GmLPAT4B transgenic plants showed more rosstte branches.To express the exogenous genes only in seeds, seed-specific promoters were studied. Through the genomic analysis one of the oleosin genes (ole8) have the target promoter. RT-PCR was done to detect the expression level in various tissues. The results showed that ole8 was only expressed in immature seeds. To confirm the function of ole8 gene promoter, the promoter was cloned and transformed into Arabidopsis. The detection of GUS showed that GUS gene expressed in seeds and cotyledon, hypocotyledonary axis of 7-days seedlings, not in other tissues and development phase. So the ole8 gene promoter is the seed-specific promoter. |
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