Establishment of an Efficient Screening System in Gene Transformation of High Oleic Acid Peanut Using AhFAD2B as a Reporter Gene
2021
Li, W. J. | Yang, L. | Zhao, C. Z. | Li, A. Q. | Li, C. S. | Wang, B. S. | Wang, X. J. | Zhao, S. Z.
During gene transformation, selection makers are important for efficient screening of the transgenic lines. The commonly used selection marker genes in plants such as neo, bar and hpt confer antibiotics resistance or herbicide resistance to the transgenic lines, which may have uncertain effects on environment and biosafety. Other selection marker genes with non-toxic products such as xylose isomerase, phosphomannose isomerase and β-glucuronidase are exogenous genes and may also have side effects on transgenic plants. Hence, endogenous and safe marker genes are desirable. Here, a new vector designated pCAMBIA2300-35S-OCS-AhFAD2B was constructed, in which the NeoR gene was replaced by AhFAD2B gene as a reporter gene. FAD2B catalyzes the formation of linoleic acid from oleic acid. High oleic acid peanut (Arachis hypogaea L.) (oleic acid content is >70% of the total fatty acids) variety Kainong1715 was used for transformation by Agrobacterium tumefaciens injection through peanut calyx tube. A total of 688 seeds were harvested, and the oleic acid content of each seed was measured using near infrared reflectance spectroscopy (NIRS). The oleic acid content of 93 seeds was found to be less than 70% indicating that these seeds had been transformed. Further validation by PCR amplification demonstrated that these seeds were true transgenic seeds. AhFAD2B as a reporter gene in peanut could greatly improve screening efficiency of transgenic seeds using NIRS. In addition, linoleic acid, the catalytic product of AhFAD2B, is the essential fatty acid for human health. This is the first report on the development of marker using endogenous gene in peanut.
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