Team:GDSYZX/proofOfConcept
Heterologous Expression Of HQT In Oryza sativa L.
We hypothesize that the constitutive promoter 35s, which could be stably expressed in all tissues, is attached to the vector and transfer into the protoplast of rice to detect the successful expression of HQT. Western Blot results show that HQT expression could be detected in the rice protoplasts transfected with 35s: : HQT, and the expression level of HQT increased with the prolongation of expression time. Thus, it can be concluded that the transferred HQT can be heterogenously expressed in rice.
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The Effect Of HQT Expression On The Content Of Chlorogenic Acid
In order to verify that HQT expression is able to improve the content of chlorogenic acid in rice protoplasts, We use HPLC technology to detect 35s::HQT transfected rice protoplasts. By comparing the results of HPLC-wild type, chlorogenic acid standard and 35s::HQT, we find that chlorogenic acid can be detected at 35s::HQT, while the expression of wild type cannot be detected. This indicate that the system we designed can successfully increase the content of chlorogenic acid in rice protoplasts.
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Endosperm Specific Expression Of HQT
Since our objective is to transfer HQT into rice seeds, we hope that HQT can be specifically expressed in rice endosperm. According to the literature we reviewed, GLUD-1 is a promoter capable of specific expression of genes in endosperm. However, due to its spatio-temporal specificity, GLud-1 cannot be expressed in protoplasts.
We detected glUD-1: : HQT system in the rice protoplast in the two dimensions of HQT protein expression and chlorogenic acid content. The results of Western Blot showed that HQT expression could not be detected in the rice protoplasts transfected with GLUD-1: : HQT; HPLC results showed that the increase of chlorogenic acid content in the rice protoplasts transfected with GLUD-1: : HQT could not be detected. Meanwhile, there was no difference between the rice protoplasts transfected with GLUD-1: : HQT and the wild-type, which were consistent with the results of previous literature investigation and our expectations. By comparing the results of 35s::HQT in STEP 1 and STEP 2, we could further verify the endosperm expression specificity of GLUD-1 promoter.
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Conclusion
We successfully expressed the HQT gene heterologously in rice protoplasts, and verified that the expression of the HQT gene can indeed increase the content of chlorogenic acid in rice. In order to express HQT specifically in rice seeds, we introduced the endosperm-specific promoter GluD-1 and confirmed the specificity of GluD-1 promoter.