农杆菌介导的葡萄胚性与非胚性愈伤组织遗传转化过程中胁迫响应基因的表达(英文)

Expression of stress response genes in EC and NEC of Vitis vinifera during Agrobacterium-mediated transformation

葡萄愈伤组织经农杆菌侵染后会产生细胞褐化和坏死现象,大大降低了转基因效率,此过程中胁迫基因发挥了重要作用。我们通过sqRT-PCR的方法研究了葡萄胚性(EC)和非胚性(NEC)愈伤组织在农杆菌侵染前、侵染30 min以及共培养3 d后PR-10,Mn-SOD,APX,IRL 5,CAT和β-1,3-glucanases基因的表达情况。EC和NEC中PR-10表达量均较高,并在侵染过程中呈上升趋势;NEC的Mn-SOD表达量没有明显变化,EC侵染30 min后Mn-SOD表达量的相对灰度值提高为侵染前的1.8倍,共培养3 d后又降低为侵染前的0.9倍;NEC的APX表达量低且比较稳定,EC在侵染前、侵染30 min和共培养3 d后APX表达量的相对灰度值分别为NEC的3.2倍、2.1倍和4.1倍;NEC中CAT表达量较低,且在转基因过程中呈现逐渐降低的趋势,EC在侵染30 min后CAT表达量的相对灰度值提高为侵染前的2.4倍;NEC中IRL 5的表达量非常低,和CAT一样在转基因过程中呈现下降趋势,而EC中该基因的表达量相对高一些;NEC的β-1,3-glucanases表达量远远高于EC,侵染前、侵染30 min和共培养3 d后其表达量的相对灰度值分别为EC的29倍、5.5倍和4.7倍。我们还研究了蛋白酶处理以后,这些基因在EC中的表达情况。结果显示:共培养3 d后进行蛋白酶处理的EC中Mn-SOD,APX,IRL 5这3个基因表达量相对灰度值降低为未处理EC的0.21、0.74和0.10,CAT和β-1,3-glucanases的表达量分别提高为未处理EC的3.7倍和8.7倍。这些结果在一定程度上说明了葡萄胚性和非胚性愈伤对于农杆菌侵染呈现不同褐化反应的潜在原因,对于我们在基因水平上研究这2种愈伤组织经过农杆菌侵染后的细胞可塑性提供了新的视角,有利于将来进一步降低愈伤褐化和细胞死亡的研究,从而提高转基因效率。

Callus of grape inoculated with Agrobacterium tumefaciens often suffer cell browning and necrosis, which greatly reduces the efficiency of transformation. Stress response genes seem to play key roles during this process. Using semi-quantitative reverse transcription-polymerase chain reaction （sqRT-PCR）, we studied the expression levels of pathogenesis-related protein 10 （PR-1O）, manganese-super-oxide dismutase （ Mn-SOD ）, ascorbate peroxidase （APX ）, isoflavone reductase-like protein 5 （ IRL 5）, catalase （CAT） and β-1, 3-glucanases genes in embryogenic callus （EC） and non-embryogenic callus （NEC） of Vitis vinifera L. at 0 min, 30 min and 3 d after inoculation with A. tumefaciens. The expression level of PR-10 gene was quite high in both NEC and EC and gradually increased during co-cultiva- tion. Mn-SOD in NEC had no obvious change, while the relative expression level of Mn-SOD in EC 30 min after inoculation increased to 1.8 times that of EC betore inoculation. At 3 dot co-cultivation, it re- duced to 0.9 times. The expression of A PX in NEC was stable and low during the process, while the ex- pression level in EC before inoculation, 30 min and 3 d after inoculation was 3.2, 2.1 and 4.1 times higher compared to the corresponding level in NEC, respectively. The expression of CAT in NEC was very low and gradually declined. The expression in EC 30 min after inoculation increased by 2.4 times compared to that before inoculation. IRL 5 presented the same decreasing tendency in NEC as CAT, and its expression was relatively high in EC. The expression of β-1,3-glucanases was much higher in NEC than EC. Before inoculation, 30 min after inoculation and 3 d after co-cultivation, the relative expression intensity of β-1,3-glucanases in NEC was 28, 4.5 and 3.7 times higher than in EC. Further more, the expression of these genes in EC treated with protease was also detected. After 3 d of co-cultivation, the expression of Mn-SOD, APX and IRL 5 in protease treated EC were reduced, but the expression of CA T and β-1,3- glucanases increased to 3.7 and 8.7 times as high as that in EC without protease treatment, respectively. The results, to a certain extent, unraveled the mechanism underlying the difference in reactions to A. tumefaciens between EC and NEC and provided a new insight into Agrobacterium-induced cell plasticity at gene level, which could be useful for strategies to reduce callus browning and cell death and thus im- prove grape transformation efficiency.