Expression of QQS gene in transgenic cassava plant: implication in starch and protein content
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Cassava, Manihot Esculenta Crantz, is a dicot woody perennial shrub that belongs to Euphorbiaceae family. Over 800 million persons globally and 250 million sub-Saharan Africans rely on the starchy root crop cassava as their staple and major daily source of calories. It is a good source of calories for human and animals because of its richness in starch (30-60% dry matter). A typical cassava-based diet provides < 30% of the minimum daily requirement (MDR) for protein and 10% –20% for iron, zinc, and pro-vitamin A. Cassava deficiency in important nutrients especially proteins (1% to 2% dry weight basis) is a major cause of death and morbidity of resources poor people in developing countries who rely exclusively on this crop for their daily calorie source. Many attempts to use both traditional and biotechnology approaches to overcome protein deficiency in cassava have been proven to be unsuccessful. Our goal is to employ modern technologies to improve the health of millions of cassava consumers through the development of bioengineered cassava with increased protein levels. Qui-Quine Starch (QQS), a novel orphan gene unique to Arabidopsis thaliana was used in this study for the development of novel transgenic cassava plants exhibiting higher protein accumulation in their tissues. QQS is fully sequenced and available in the Genbank (GenBank#: NM_113975). This gene acts as a transcription factor and is known to impart carbon and nitrogen repartition into plant and therefore causing variations in protein, carbohydrate and even oil accumulation when expressed heterologously in transgenic plants. We expressed QQS in transgenic cassava plant via Agrobacterium-mediated genetic transformation with the goal to increase protein accumulation and study the eventual variations in plant morphology and biochemical profiles of other metabolites such as carbohydrates. A total of 10 transgenic cassava lines stably expressing QQS were regenerated and all well established in soil and only two lines (20%) showing malformed phenotype. Line R7 F) has the best growth vigor. Traditional PCR demonstrated that QQS gene was integrated in the genome of all the lines. RT-PCR data showed that the QQS gene is differentially expressed and that its transcript levels varied remarkably between shoot and root biomass of each transgenic line. Results of the nutritional profiles have shown that the expression of QQS gene into cassava plant increased leaf protein by 1.36% in line R’’’ (LA) L2 and root protein by 17.02% for the same line compared to their wild-type control plants and non-QQS expressers. Moreover, leaf soluble total carbohydrate increased by 51.76% in line R’’’ (G) L2 and root soluble total carbohydrate increased by 46.75% in line R7 (F). Regression analysis established no correlation between QQS transcript levels, carbohydrate, and protein content for transgenic root and shoot samples. Novel functionality of QQS to increase starch content in transgenic biomass is demonstrated. No change in the content of specific amino acids was observed among lines and plant parts. In addition, QQS expression revealed an increase in biomass, plant vigor, early in-vitro mini-tubers production for line R7 (F). However, it will be very critical to validate these observed changes in nutritional profiles of these young cassava plants expressing QQS by biochemically profiling these transgenic cassava plants at maturity once established in the field. Once validated in model cv60444, the transferability of this technology to consumer’s preferred cassava plant parts (tuberous roots and leaves) should be attempted.